Effective vaccination against porcine reproductive and respiratory syndrome (prrs) virus prior to weaning

ABSTRACT

The invention provides isolated polynucleotide molecules that comprise a DNA sequence encoding an infectious RNA sequence encoding a genetically-modified North American PRRS virus, methods to make it and related polypeptides, polynucleotides, and various components. Vaccines comprising the genetically modified virus and polynucleotides and a diagnostic kit to distinguish between naturally infected and vaccinated animals are also provided.

FIELD OF THE INVENTION

The present invention is in the field of animal health and is directedto infectious cDNA clones of positive polarity RNA viruses, novel RNAviruses and modified live forms thereof, and the construction ofvaccines, in particular, swine vaccines, using such cDNA clones. Moreparticularly, the present invention also provides for the safe and earlyvaccination of piglets prior to weaning, including from immediatelyafter birth (i.e. only 1 day of age or less) to two weeks of age, at alltimes optionally in combination with multivalent combination swinevaccines, such as bivalent PRRSV/Mycoplasma hyopneumoniae (M.hyo)vaccines, bivalent PRRSV/Porcine Circovirus type 2 (PCV2) vaccines, andtrivalent PRRSV/M.hyo/PCV2 vaccines, or simply as a monovalent PRRSVvaccine. Early vaccination against PRRS under such conditions providesan early onset of protective immunity, that arises no later than about14 days after vaccination, i.e. at Day 15 following vaccination on Day 1of life, Day 21 following vaccination on Day 7 of life, and no laterthan about Day 28 following vaccination on Day 14 of life. Although thepresent specification provides for numerous constructs of the “P129strain” of North American PRRS virus (see PCT/IB2011/055003 and U.S.Pat. No. 6,500,662), which are highly effective as vaccines, includingfor such early and safe use, it has been determined that such earlyonset of protective immunity (i.e. about 2 weeks following immunizingvaccination given as early as Day 1 after birth), is also applicable touse of other North American and European PRRS strains, such as thosedescribed in U.S. Pat. No. 5,476,778, U.S. Pat. No. 5,846,805, U.S. Pat.No. 6,380,376, U.S. Pat. No. 6,982,160 and U.S. Pat. No. 6,197,310.

BACKGROUND OF THE INVENTION

Porcine reproductive and respiratory syndrome (PRRS) is characterized byabortions, stillbirths, and other reproductive problems in sows andgilts, as well as respiratory disease in young pigs. The causative agentis the PRRS virus (PRRSV), a member of the family Arteriviridae and theorder Nidovirales. The nidoviruses are enveloped viruses having genomesconsisting of a single strand of positive polarity RNA. The genomic RNAof a positive-stranded RNA virus fulfills the dual role in both storageand expression of genetic information. No DNA is involved in replicationor transcription in Nidoviruses. The non-structural proteins aretranslated directly from the genomic RNA of nidoviruses as largepolyproteins and subsequently cleaved by viral proteases into discreetfunctional proteins. A 3′-coterminal nested set of subgenomic RNAs(sgRNAs) is synthesized from the genome and are used as messenger RNAsfor translation of the structural proteins. The reproduction ofnidoviral genomic RNA is thus a combined process of genome replicationand sgRNA synthesis.

In the late 1980's, two distinct genotypes of the virus emerged nearlysimultaneously, one in North America and another in Europe. PRRS virusis now endemic in nearly all swine producing countries, and isconsidered one of the most economically important diseases affecting theglobal pork industry. Additionally, highly virulent genotypes have beenisolated in China and surrounding countries, and such genotypes aregenerally related to North American genotypes.

Despite significant advances in understanding the biology of PRRSV,control of the virus remains difficult. Vaccination of animals in thefield has proven to be largely ineffective. PRRS commonly re-emerges inimmunized herds, and most on-farm PRRSV vaccination campaigns ultimatelyfail to control the disease.

Without being limited as to theory, infection of pigs with wild typePRRSV or their vaccination with a live attenuated form of this pathogenunfortunately only elicits an exuberant production of non-neutralizingantibodies. During this time interval, for example, only limitedquantities of interferon (IFN)-γ (secreting cells are generated. Thus,PRRSV seems to inherently stimulate an imbalanced immune responsedistinguished by consistently abundant humoral (antibody-based)immunity, and a variable and limited but potentially protective T helper(Th) 1—like IFN-γ response. One characteristic of PRRSV infection thatmost likely contributes to the imbalanced development of adaptiveimmunity is the lack of an adequate innate immune response. Usually,virus-infected cells secrete type I interferon “IFN” (including IFN-αand IFN-β), which protects neighboring cells from infection. Inaddition, the released type I IFN interacts with a subset of naïve Tcells to promote their conversion into virus-specific type II IFN(IFN-γ) secreting cells. In contrast, the IFN-α response of pigs toPRRSV exposure is nearly non-existent. Such inefficient stimulation ofIFN-α production by a pathogen would be expected to have a significantimpact on the nature of the host's adaptive immune response, since IFN-αup-regulates IFN-γ gene expression. Accordingly, the former cytokinecontrols the dominant pathway that promotes the development of adaptiveimmunity, namely, T cell-mediated IFN-γ responses and peak antiviralimmune defenses.

In this regard, it has become evident that a probable link betweeninnate and adaptive immunity in viral infections occurs through aspecial type of dendritic cell which has the ability to produce largeamounts of type I interferon, and which plays a critical role in thepolarization of T-cell function. Specifically, an infrequent butremarkable type of dendritic cell, the plasmacytoid dendritic cell(PDC), also known as a natural IFN-α/β-producing cell, plays a criticalrole in anti-viral immunity by means of their ability to cause naïve Tcells to differentiate into IFN-γ secreting cells. Although rare, thePDC are enormously potent producers of IFN-α, with each cell beingcapable of producing 3-10 pg of IFN-α in response to virus. In contrast,monocytes produce 5- to 10-fold less IFN-α on a per cell basis. Thephenotype and some biological properties of porcine PDC have beendescribed (Summerfield et al., 2003, Immunology 110:440). Recent studieshave determined that PRRSV does not stimulate porcine PDCs to secreteIFN-α (Calzada et al., 2010, Veterinary Immunology and Immunopathology135:20).

This fact, in combination with the observation that exogenously addedIFN-α at the time of vaccination has been found to improve the intensityof the PRRSV-specific IFN-γ response (W. A. Meier et al., Vet. Immunol.Immunopath. 102, pp 299-314, 2004), highlights the critical role thatIFN-α plays during the infection of pigs with this virus. Given theapparent critical role of IFN-α on the development of protectiveimmunity, it is important to determine the ability of different PRRSvirus stocks to stimulate and/or inhibit the production of IFN-α.Accordingly, there is a pressing need for new and improved modified livevaccines to protect against PRRS. As described below, it is clear thatviruses derived from the novel infectious cDNA clone, pCMV-S-P129-PK,and others, have a different phenotype than either the wild-type P129virus or two commercially available modified live PRRS vaccines. Withoutbeing limited as to theory, the present invention provides for vaccinesthat facilitate cell-based immune response against the virus, and definea new and effective generation of PRRS vaccines.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention provides an isolatedpolynucleotide molecule including a DNA sequence encoding an infectiousRNA molecule encoding a PRRS virus that is genetically modified suchthat, as a vaccine, it elicits an effective immunoprotective responseagainst the PRRS virus in porcine animals. In certain aspects, theinvention provides for a DNA sequence as set forth herein including SEQID NO.:1, SEQ ID NO.:2, SEQ ID NO.:3, SEQ ID NO.:4, or SEQ ID: NO:6, ora sequence having at least 70% identity thereto, preferably 80% identitythereto, and more preferably 85%, 90%, 95%, 96%, 97%, 98% or 99%identity thereto.

In certain embodiments, the invention provides for a plasmid thatincludes an isolated polynucleotide molecule as set forth herein and apromoter capable of transcribing the polynucleotide molecule in asuitable host cell. In another embodiment, the North American or ChinesePRRS encoding sequence of the plasmid herein further encodes one or moredetectable heterologous antigenic epitopes. The present inventionprovides for a transfected host cell that includes the plasmid set forthherein.

In another aspect, the present invention provides for a vaccine forprotecting a porcine animal from infection by a PRRS virus. The vaccinemay include a North American or Chinese PRRS virus encoded by aninfectious RNA molecule, the infectious RNA molecule, or a plasmid, eachof which are encoded by the isolated polynucleotide molecule as setforth herein. In yet another aspect, the vaccine includes a viral vectorincluding the polynucleotide herein. The vaccine set forth herein mayoptionally include a vaccine carrier acceptable for veterinary use. Inone important aspect, the vaccine has a decreased interferon-αinhibitory effect as compared to wild-type P129 PRRS virus (see ATCC203488, 203489, U.S. Pat. No. 6,500,662).

In one embodiment, the present invention provides for diagnostic kitincluding polynucleotide molecules which distinguish (a so-called DIVAtest) between porcine animals naturally infected with a field strain ofa PRRS virus and porcine animals vaccinated with the modified livevaccine set forth herein.

In other embodiments, the invention provides for a method of protectinga porcine animal from infection with a strain of PRRS virus includingadministering to the animal an immunogenically protective amount of thevaccine of the claims set forth herein.

Further and preferred embodiments of the invention include an isolatedPorcine Reproductive and Respiratory Syndrome Virus (PRRS), or apolynucleotide sequence encoding therefor, wherein the protein encodedby ORF1a is selected from a group consisting of those that contain anyof the following amino acid sequences, wherein the underlined residuesare believed to be novel: AMANVYD (SEQ ID NO: 9); IGHNAVM (SEQ ID NO:12); TVPDGNC (SEQ ID NO: 15); CWWYLFD (SEQ ID NO: 18); HGVHGKY (SEQ IDNO: 21); AAKVDQY (SEQ ID NO: 24); PSATDTS (SEQ ID NO: 27); LNSLLSK (SEQID NO: 30); APMCQDE (SEQ ID NO: 33); CAPTGMD (SEQ ID NO: 36); PKVAKVS(SEQ ID NO: 39); AGEIVGV (SEQ ID NO: 42); ADFNPEK (SEQ ID NO: 45); andQTPILGR (SEQ ID NO: 48). In a further preferred embodiment of theinvention, the invention provides an isolated North American or ChinesePRRS that contain any of the above-identified sequences within theprotein encoded from ORF1a, including any combinations (2, 3, 4 . . . upto 17) of these identified sequences.

The invention further provides for an isolated Porcine Reproductive andRespiratory Syndrome Virus (PRRS) wherein the protein thereof encoded byORF1a is selected from a group consisting of those amino acid sequencesthat contain any of: ANV (see SEQ ID NO: 9); HNA (see SEQ ID NO: 12);PDG (see SEQ ID NO: 15); WYL (see SEQ ID NO: 18); VHG (see SEQ ID NO:21); KVD (see SEQ ID NO: 24); ATD (see SEQ ID NO: 27); SLL (see SEQ IDNO: 30); MCQ (see SEQ ID NO: 33); PTG (see SEQ ID NO: 36); VAK (see SEQID NO: 39); EIV (see SEQ ID NO: 42); FNP (see SEQ ID NO: 45); and PIL(see SEQ ID NO: 48), including any combinations (2, 3, 4 . . . up to 17)of these identified sequences.

In a further preferred embodiment, the invention provides an isolatedNorth American or Chinese PRRS wherein, irrespective of the identity ofany other specific nucleotide or amino acid sequence positions at anypoint in a polynucleotide encoding the virus or the proteins encodedtherefrom, the ORF1a virus protein contains:

(a) any of the following specific amino acids in the specifiedsequences,an amino acid N within the amino acid sequence ANV (see SEQ ID NO: 9);an amino acid N within the amino acid sequence HNA (see SEQ ID NO: 12);an amino acid D within the amino acid sequence PDG (see SEQ ID NO: 15);an amino acid Y within the amino acid sequence WYL (see SEQ ID NO: 18);an amino acid H within the amino acid sequence VHG (see SEQ ID NO: 21);an amino acid V within the amino acid sequence KVD (see SEQ ID NO: 24);an amino acid T within the amino acid sequence ATD (see SEQ ID NO: 27);an amino acid L within the amino acid sequence SLL (see SEQ ID NO: 30).an amino acid C within the amino acid sequence MCQ (see SEQ ID NO: 33);an amino acid T within the amino acid sequence PTG (see SEQ ID NO: 36);an amino acid A within the amino acid sequence VAK (see SEQ ID NO: 39);an amino acid I within the amino acid sequence EIV (see SEQ ID NO: 42);an amino acid N within the amino acid sequence FNP (see SEQ ID NO: 45);andamino acid I within the amino acid sequence PIL (see SEQ ID NO: 48), toinclude any combinations (2, 3, 4 . . . up to 17) of these identifiedsequences, or(b) contains said specific underlined single amino acids in thespecified 3-residue ORF1a peptide sequences of any other North Americanor Chinese PRRS viruses that correspond to the 3-residue sequences asspecified above, taking into account that said other specific 3-residueamino acid sequences may show one or two additional amino sequencechanges, but still be recognized as corresponding to the sequencesspecified above. For the purposes of this embodiment of the invention,“corresponding” means that the relative sequences can be optimallyaligned using a BLOSUM algorithm as described in Henikoff et al. ProcNatl. Acad. Sci., USA, 89, pp. 10915-10919, 1992.

In a further preferred embodiment of the invention, an isolated PorcineReproductive and Respiratory Syndrome Virus (PRRS) is provided whereinthe protein thereof encoded by ORF1a has an amino acid sequence thatcontains one or more of variations (a), (b), (c) and (d), wherein eachsaid variation is defined as follows:

variation (a),an amino acid N within the amino acid sequence ANV (see SEQ ID NO: 9);an amino acid N within the amino acid sequence HNA (see SEQ ID NO: 12);an amino acid D within the amino acid sequence PDG (see SEQ ID NO: 15),an amino acid Y within the amino acid sequence WYL (see SEQ ID NO: 18);an amino acid H within the amino acid sequence VHG (see SEQ ID NO: 21),or any subset of variation (a);variation (b),an amino acid V within the amino acid sequence KVD (see SEQ ID NO: 24);an amino acid T within the amino acid sequence ATD (see SEQ ID NO: 27);an amino acid L within the amino acid sequence SLL (see SEQ ID NO: 30).an amino acid C within the amino acid sequence MCQ (see SEQ ID NO: 33),or any subset of variation (b);variation (c),an amino acid T within the amino acid sequence PTG (see SEQ ID NO: 36);an amino acid A within the amino acid sequence VAK (see SEQ ID NO: 39),or any subset of variation (c); andvariation (d),an amino acid I within the amino acid sequence EIV (see SEQ ID NO: 42);an amino acid N within the amino acid sequence FNP (see SEQ ID NO: 45);and amino acid I within the amino acid sequence PIL (see SEQ ID NO: 20),or any subset of variation (d) thereof.

Such PRRS viruses may further contain two or more of the five amino acidsequences identified in variation (a), and/or two or more of the fouramino acid sequences identified in variation (b), and/or the two aminoacid sequences identified in variation (c), and/or two or more of thethree amino acid sequences identified in variation (d).

The present invention also provides a plasmid capable of directlytransfecting a suitable host cell and expressing a Porcine Reproductiveand Respiratory Syndrome Virus (PRRS) from the suitable host cell sotransfected, which plasmid comprises: (a) a DNA sequence encoding aninfectious RNA molecule encoding the PRRS virus, and (b) a promotercapable of transcribing said infectious RNA molecule, wherein theprotein encoded by ORF1a of said virus has an amino acid sequence thatcontains:

(1) an amino acid N within the amino acid sequence ANV (see SEQ ID NO:9);an amino acid N within the amino acid sequence HNA (see SEQ ID NO: 12);an amino acid D within the amino acid sequence PDG (see SEQ ID NO: 15),an amino acid Y within the amino acid sequence WYL (see SEQ ID NO: 18);an amino acid H within the amino acid sequence VHG (see SEQ ID NO: 21),or any subset thereof; and/or(2) an amino acid V within the amino acid sequence KVD (see SEQ ID NO:24);an amino acid T within the amino acid sequence ATD (see SEQ ID NO: 27);an amino acid L within the amino acid sequence SLL (see SEQ ID NO: 30).an amino acid C within the amino acid sequence MCQ (see SEQ ID NO: 33),or any subset thereof; and/or(3) an amino acid T within the amino acid sequence PTG (see SEQ ID NO:36);an amino acid A within the amino acid sequence VAK (see SEQ ID NO: 39),or any subset thereof; and/or(4) an amino acid I within the amino acid sequence EIV (see SEQ ID NO:42);an amino acid N within the amino acid sequence FNP (see SEQ ID NO: 45);and amino acid I within the amino acid sequence PIL (see SEQ ID NO: 48),or any subset thereof.

It will be appreciated that ORF1a encodes a polyprotein comprisingprotease function, and ORF1b encodes a polyprotein comprising replicase(RNA polymerase) and helicase functions. Additional informationconcerning the functions for proteins encoded from various ORFs (openreading frames) of PRRS may be found, for example, in U.S. Pat. No.7,132,106. See also U.S. Pat. No. 7,544,362 in regard of function ofORF7, and other open reading frames. As would be appreciated in the art,the ORF1-encoded proteins are expected to have additional functions,known and unknown, and the novel amino acid changes useful in thepractice of the present invention are not limited via their effects onany one specific function of the ORF1-encoded proteins.

In further preferred embodiments, said plasmid contains a promoter thatis a eukaryotic promoter capable of permitting a DNA launch in targetedeukaryotic cells, or a prokaryotic or phage promoter capable ofdirecting in vitro transcription of the plasmid. The invention similarlyprovides a method of generating a PRRS virus, which method comprisestransfecting a suitable host cell with an appropriate plasmid andobtaining PRRS virus generated by the transfected cell.

Accordingly, in a specific and preferred embodiment, the inventionprovides an isolated polynucleotide molecule comprising a DNA sequenceencoding an infectious RNA molecule encoding a North American PRRSvirus, wherein said DNA sequence is selected from the group consistingof:

(a) SEQ ID NO:6;

(b) a sequence that has at least 85% identity to the DNA sequence of (a)wherein the protein encoded by ORF1a thereof has an amino acid sequencethat contains: from group (b)(1)an amino acid N within the amino acid sequence ANV (see SEQ ID NO: 9);an amino acid N within the amino acid sequence HNA (see SEQ ID NO: 12);an amino acid D within the amino acid sequence PDG (see SEQ ID NO: 15),an amino acid Y within the amino acid sequence WYL (see SEQ ID NO: 18);an amino acid H within the amino acid sequence VHG (see SEQ ID NO: 21),or any subset thereof; and/or from group (b) (2)an amino acid V within the amino acid sequence KVD (see SEQ ID NO: 24);an amino acid T within the amino acid sequence ATD (see SEQ ID NO: 27);an amino acid L within the amino acid sequence SLL (see SEQ ID NO: 30).an amino acid C within the amino acid sequence MCQ (see SEQ ID NO: 33),or any subset thereof; and/orfrom group (b)(3)an amino acid T within the amino acid sequence PTG (see SEQ ID NO: 36);an amino acid A within the amino acid sequence VAK (see SEQ ID NO: 39),or any subset thereof; and/orfrom group (b)(4)an amino acid I within the amino acid sequence EIV (see SEQ ID NO: 42);an amino acid N within the amino acid sequence FNP (see SEQ ID NO: 45);and amino acid I within the amino acid sequence PIL (see SEQ ID NO: 20),or any subset thereof; and(c) a DNA sequence that hybridizes to the complement of a DNA sequenceof (a) or (b) under highly stringent conditions which comprisehybridization to filter bound DNA in 0.5 M NaHPo4, 7% SDS, 1 mM EDTA at65 degrees C., and washing in 0.1 SSC/0/1% SDS at 68 degrees C.

The invention also provides for host cells transfected withpolynucleotide molecules and provides vaccines for protecting a porcineanimal against infection by a PRRS virus, which vaccine comprises: (a) agenetically modified North American PRRS virus encoded by suchaforementioned polynucleotide molecules, or (b) said infectiousmolecule, or (c) said polynucleotide molecule in the form of a plasmid,or (d) a viral vector comprising said polynucleotide molecule, whereinthe PRRS virus is able to elicit an effective immunoprotective responseagainst infection by PRRS virus, in an amount effective to produceimmunoprotection against infection, and a carrier suitable forveterinary use.

The invention also provides RNA polynucleotide sequences correspondingto (i.e. by having complementary base coding sequences):

(a) the DNA sequence of SEQ ID NO:6;(b) a DNA sequence that has at least 85% identity to the DNA sequence of(a) wherein the protein encoded by ORF1a thereof has an amino acidsequence that contains any of the following, and any combination of anyof the following:an amino acid N within the amino acid sequence ANV (see SEQ ID NO: 9);an amino acid N within the amino acid sequence HNA (see SEQ ID NO: 12);an amino acid D within the amino acid sequence PDG (see SEQ ID NO: 15),an amino acid Y within the amino acid sequence WYL (see SEQ ID NO: 18);an amino acid H within the amino acid sequence VHG (see SEQ ID NO: 21),an amino acid V within the amino acid sequence KVD (see SEQ ID NO: 24);an amino acid T within the amino acid sequence ATD (see SEQ ID NO: 27);an amino acid L within the amino acid sequence SLL (see SEQ ID NO: 30).an amino acid C within the amino acid sequence MCQ (see SEQ ID NO: 33),an amino acid T within the amino acid sequence PTG (see SEQ ID NO: 36);an amino acid A within the amino acid sequence VAK (see SEQ ID NO: 39),an amino acid I within the amino acid sequence EIV (see SEQ ID NO: 42);an amino acid N within the amino acid sequence FNP (see SEQ ID NO: 45);and amino acid I within the amino acid sequence PIL (see SEQ ID NO: 20),or(c) a DNA sequence that hybridizes to the complement of a DNA sequenceof (a) or (b) under highly stringent conditions which comprisehybridization to filter bound DNA in 0.5 M NaHPo4, 7% SDS, 1 mM EDTA at65 degrees C., and washing in 0.1 SSC/0/1% SDS at 68 degrees C.

Accordingly, the invention also provides diagnostic kits comprisingpolynucleotide molecules which distinguish between porcine animalsnaturally infected with a field strain of a PRRS virus and porcineanimals vaccinated with the vaccines of the invention, which vaccines(viruses) preferably evidence a a decreased interferon-α inhibitoryeffect as compared to wild-type P129 PRRS virus (SEQ ID NO:5).

BRIEF DESCRIPTION OF THE TABLES AND FIGURES

Table 1 shows infectious cDNA clones and the corresponding viruses thatwere derived by transfection into PK-9 cells.

Table 2 shows the interferon-α inhibitory effect of wild-type PRRS virusand derivatives adapted to growth in cell culture.

Table 3 delineates the interferon-α inhibitory effect of wild-type PRRSvirus P129 and its genetically engineered derivatives adapted to grow inCD163-expressing PK-9 cells.

Table 4 shows decreased interferon-α inhibitory effect of the P129-PK-FLand P129-PK-d43/44 viruses as compared to the wild-type P129 virus andthe PRRS Ingelvac vaccines.

Table 5 depicts the design of a study conducted to evaluate the safetyand efficacy of vaccine viruses.

Table 6 shows all nucleotide differences and resulting amino aciddifferences between P129 passage 0 and P129-PK-FL passage 17, by genomeposition.

Table 7 shows a summary of nucleotide and amino acid differences betweenP129 passage 0 and P129-PK-FL passage 17, by viral protein.

Table 8 shows all nucleotide differences and resulting amino aciddifferences between the PRRSV genomes found in infectious cDNA clonespCMV-S-P129 and pCMV-S-P129-PK17-FL, by genome position.

Tables 9 and 10 show amino acid changes contributing to the phenotype ofthe Passage 52 virus (SEQ ID NO:6).

Table 11 shows numbers of pigs with clinical signs following vaccinationat one day of age with a modified live PRRSV vaccine.

Table 12 shows serum mean titers following vaccination at one day of agewith a modified live PRRSV vaccine.

Table 13 shows percent lung lesions following a challenge of 7-week oldpigs previously vaccinated at one day of age with a modified live PRRSVvaccine.

Table 14 shows percent lung lesions following a challenge of 18-week oldpigs previously vaccinated at one day of age with a modified live PRRSVvaccine.

Table 15 shows percent lung lesions following a challenge of 26-week oldpigs previously vaccinated at one day of age with a modified live PRRSVvaccine.

Table 16 shows percent lung lesions following a challenge of 5-week oldpiglets previously vaccinated with a modified live PRRSV vaccine.

FIG. 1 shows rectal temperatures post-vaccination.

FIG. 2 shows rectal temperatures post-challenge with virulent PRRSVNADC20.

FIG. 3 shows body weights post-vaccination and post-challenge.

FIG. 4 shows post-challenge data for percentage of lungs with PRRSlesions.

FIG. 5 shows post-challenge lung assessment scores (LAS) for severity oflesions observed.

FIG. 6 is a histogram that depicts the anti-PRRSV antibody levels inserum post-vaccination and post-challenge (ELISA S/P ratios).

FIG. 7 is a graphical representation of post-challenge virus load inserum (log TCID50/ml on PAM cells)

FIG. 8 is a pictorial representation of the methods employed forobtaining the vaccines including SEQ ID NO:1 through SEQ ID NO:6, asdisclosed herein.

BRIEF DESCRIPTION OF THE MAJOR SEQUENCES

SEQ ID NO:1 provides the P129-PK-FL passage 17 complete genome.

SEQ ID NO:2 provides the P129-PK-d43/44 passage 17 complete genome.

SEQ ID NO:3 provides the P129-PK-FL passage 24 complete genome.

SEQ ID NO:4 provides the P129-PK-d43/44 passage 34 complete genome.

SEQ ID NO:5 provides the P129 passage 0 complete genome.

SEQ ID NO:6 provides the P129 passage 52 complete genome.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

“North American PRRS virus” means any PRRS virus having geneticcharacteristics associated with a North American PRRS virus isolate,such as, but not limited to the PRRS virus that was first isolated inthe United States around the early 1990's (see, e.g., Collins, J. E., etal., 1992, J. Vet. Diagn. Invest. 4:117-126); North American PRRS virusisolate MN-1b (Kwang, J. et al., 1994, J. Vet. Diagn. Invest.6:293-296); the Quebec LAF-exp91 strain of PRRS (Mardassi, H. et al.,1995, Arch. Virol. 140:1405-1418); and North American PRRS virus isolateVR 2385 (Meng, X.-J et al., 1994, J. Gen. Virol. 75:1795-1801). Geneticcharacteristics refer to genomic nucleotide sequence similarity andamino acid sequence similarity shared by North American PRRS virusstrains. Chinese PRRS virus strains generally evidence about 80-93%nucleotide sequence similarity with North American strains.

“European PRRS virus” refers to any strain of PRRS virus having thegenetic characteristics associated with the PRRS virus that was firstisolated in Europe around 1991 (see, e.g., Wensvoort, G., et al., 1991,Vet. Q. 13:121-130). “European PRRS virus” is also sometimes referred toin the art as “Lelystad virus”.

“An effective immunoprotective response”, “immunoprotection”, and liketerms, for purposes of the present invention, mean an immune responsethat is directed against one or more antigenic epitopes of a pathogen soas to protect against infection by the pathogen in a vaccinated animal.For purposes of the present invention, protection against infection by apathogen includes not only the absolute prevention of infection, butalso any detectable reduction in the degree or rate of infection by apathogen, or any detectable reduction in the severity of the disease orany symptom or condition resulting from infection by the pathogen in thevaccinated animal as compared to an unvaccinated infected animal. Aneffective immunoprotective response can be induced in animals that havenot previously been infected with the pathogen and/or are not infectedwith the pathogen at the time of vaccination. An effectiveimmunoprotective response can also be induced in an animal alreadyinfected with the pathogen at the time of vaccination.

A genetically modified PRRS virus is “attenuated” if it is less virulentthan its unmodified parental strain. A strain is “less virulent” if itshows a statistically significant decrease in one or more parametersdetermining disease severity. Such parameters may include level ofviremia, fever, severity of respiratory distress, severity ofreproductive symptoms, or number or severity of lung lesions, etc.

“Host cell capable of supporting PRRS virus replication” means a cellwhich is capable of generating infectious PRRS when infected with avirus of the invention. Such cells include porcine cells of themonocyte/macrophage lineage such as porcine alveolar macrophage cellsand derivatives, MA-104 monkey kidney cells and derivatives such asMARC-145 cells, and cells transfected with a receptor for the PRRSvirus. The term “host cell capable of supporting PRRS virus replication”may also include cells within a live pig.

“Open reading frame”, or “ORF”, as used herein, means the minimalnucleotide sequence required to encode a particular PRRS virus proteinwithout an intervening stop codon.

“Porcine” and “swine” are used interchangeably herein and refer to anyanimal that is a member of the family Suidae such as, for example, apig. The term “PRRS virus”, as used herein, unless otherwise indicated,means any strain of either the North American or European PRRS viruses.

“PRRS” encompasses disease symptoms in swine caused by a PRRS virusinfection. Examples of such symptoms include, but are not limited to,fever, abortion in pregnant females, respiratory distress, lung lesions,loss of appetite, and mortality in young pigs. As used herein, a PRRSvirus that is “unable to produce PRRS” refers to a virus that can infecta pig, but which does not produce any disease symptoms normallyassociated with a PRRS infection in the pig.

PRRSV “N protein” or “ORF7” as used herein is defined as a polypeptidethat is encoded by ORF7 of both the European and North Americangenotypes of PRRS virus. Examples of specific isotypes of N proteinwhich are currently known are the 123 amino acid polypeptide of theNorth American PRRS prototype isolate VR2322 reported in Genbank byAccession numbers PRU87392, and the 128 residue N protein of Europeanprototype PRRS isolate Lelystad reported in Genbank Accession numberA26843.

“PRRSV N protein NLS-1 region” or “PRRSV ORF7 NLS-1 region” refers to a“pat4” or “nuc1” nuclear localization signal (Nakai & Kanehisa, 1992;Rowland & Yoo, 2003) containing four continuous basic amino acids(lysine or arginine), or three basic residues and a histidine orproline, located within about the first 15 N-terminal residues of themature N protein. By way of example the VR2332 NLS-1 region sequence isKRKK and is located at residues 9-12, while the Lelystad isolatesequence is KKKK and is located at residues 10-13 of the N protein.

“PRRSV N protein NLS-2 region” or “PRRSV ORF7 NLS-2 region” refers to asecond nuclear localization signal within the N protein that can takeone of two forms. In North American PRRS viruses NLS-2 has a patternwhich we have designated as the “pat8” motif, which begins with aproline followed within three residues by a five residue sequencecontaining at least three basic residues (K or R) out of five (a slightmodification of the “pat7” or “nuc2” motif described by Nakai &Kanehisa, 1992; Rowland & Yoo, 2003). By way of example such a sequenceis located at N protein residues 41-47 of the North American PRRSVisolate VR2332, and is represented by the sequence P . . . K In EuropeanPRRS viruses NLS-2 has a “pat4” or “nuc1” motif, which is a continuousstretch of four basic amino acids or three basic residues associatedwith histidine or proline (Nakai & Kanehisa, 1992; Rowland & Yoo, 2003).The NLS-2 of the European PRRSV isolate Lelystad is located at residues47-50 and is represented by the sequence K.K

“PRRSV N protein NoLS region” or “PRRSV ORF7 NoLS region” refers to anucleolar localization signal having a total length of about 32 aminoacids and incorporating the NLS-2 region near its amino terminus. By wayof example the VR2332 NoLS region sequence is located at residues 41-72and is represented by the sequence P . . . R (Rowland & Yoo, 2003) andthe corresponding Lelystad isolate sequence is located at residues 42-73and is represented by the sequence P.R.

“Transfected host cell” means practically any host cell which asdescribed in U.S. Pat. No. 5,600,662 when transfected with PRRS virusRNA can produce a at least a first round of PRRS virions.

An “infectious DNA molecule”, for purposes of the present invention, isa DNA molecule that encodes the necessary elements to supportreplication, transcription, and translation into a functional virionfrom a suitable host cell.Likewise, an “isolated polynucleotide molecule” refers to a compositionof matter comprising a polynucleotide molecule of the present inventionpurified to any detectable degree from its naturally occurring state, ifany.

For purposes of the present invention, the nucleotide sequence of asecond polynucleotide molecule (either RNA or DNA) is “homologous” tothe nucleotide sequence of a first polynucleotide molecule, or has“identity” to said first polynucleotide molecule, where the nucleotidesequence of the second polynucleotide molecule encodes the samepolyaminoacid as the nucleotide sequence of the first polynucleotidemolecule as based on the degeneracy of the genetic code, or when itencodes a polyaminoacid that is sufficiently similar to thepolyaminoacid encoded by the nucleotide sequence of the firstpolynucleotide molecule so as to be useful in practicing the presentinvention. Homologous polynucleotide sequences also refers to sense andanti-sense strands, and in all cases to the complement of any suchstrands. For purposes of the present invention, a polynucleotidemolecule is useful in practicing the present invention, and is thereforehomologous or has identity, where it can be used as a diagnostic probeto detect the presence of PRRS virus or viral polynucleotide in a fluidor tissue sample of an infected pig, e.g. by standard hybridization oramplification techniques. Generally, the nucleotide sequence of a secondpolynucleotide molecule is homologous to the nucleotide sequence of afirst polynucleotide molecule if it has at least about 70% nucleotidesequence identity to the nucleotide sequence of the first polynucleotidemolecule as based on the BLASTN algorithm (National Center forBiotechnology Information, otherwise known as NCBI, (Bethesda, Md., USA)of the United States National Institute of Health). In a specificexample for calculations according to the practice of the presentinvention, reference is made to BLASTP 2.2.6 [Tatusova TA and TL Madden,“BLAST 2 sequences—a new tool for comparing protein and nucleotidesequences.” (1999) FEMS Microbiol Lett. 174:247-250.]. Briefly, twoamino acid sequences are aligned to optimize the alignment scores usinga gap opening penalty of 10, a gap extension penalty of 0.1, and the“blosum62” scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad.Sci. USA 89:10915-10919. 1992). The percent identity is then calculatedas: Total number of identical matches ×100/divided by the length of thelonger sequence+number of gaps introduced into the longer sequence toalign the two sequences.

Preferably, a homologous nucleotide sequence has at least about 75%nucleotide sequence identity, even more preferably at least about 80%,85%, 90% 95%, 96%, 97%, 98% and 99% nucleotide sequence identity. Sincethe genetic code is degenerate, a homologous nucleotide sequence caninclude any number of “silent” base changes, i.e. nucleotidesubstitutions that nonetheless encode the same amino acid.

A homologous nucleotide sequence can further contain non-silentmutations, i.e. base substitutions, deletions, or additions resulting inamino acid differences in the encoded polyaminoacid, so long as thesequence remains at least about 70% identical to the polyaminoacidencoded by the first nucleotide sequence or otherwise is useful forpracticing the present invention. In this regard, certain conservativeamino acid substitutions may be made which are generally recognized notto inactivate overall protein function: such as in regard of positivelycharged amino acids (and vice versa), lysine, arginine and histidine; inregard of negatively charged amino acids (and vice versa), aspartic acidand glutamic acid; and in regard of certain groups of neutrally chargedamino acids (and in all cases, also vice versa), (1) alanine and serine,(2) asparagine, glutamine, and histidine, (3) cysteine and serine, (4)glycine and proline, (5) isoleucine, leucine and valine, (6) methionine,leucine and isoleucine, (7) phenylalanine, methionine, leucine, andtyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10)and for example tyrosine, tyrptophan and phenylalanine. Amino acids canbe classified according to physical properties and contribution tosecondary and tertiary protein structure. A conservative substitution isrecognized in the art as a substitution of one amino acid for anotheramino acid that has similar properties. Exemplary conservativesubstitutions may be found in WO 97/09433, page 10, published Mar. 13,1997 (PCT/GB96/02197, filed Sep. 6, 1996. Alternatively, conservativeamino acids can be grouped as described in Lehninger, (Biochemistry,Second Edition; Worth Publishers, Inc. NY: N.Y. (1975), pp. 71-77).Additional suitable conservative changes and the application thereof aredescribed below.

Homologous nucleotide sequences can be determined by comparison ofnucleotide sequences, for example by using BLASTN, above. Alternatively,homologous nucleotide sequences can be determined by hybridization underselected conditions. For example, the nucleotide sequence of a secondpolynucleotide molecule is homologous to SEQ ID NO:1 (or any otherparticular polynucleotide sequence) if it hybridizes to the complementof SEQ ID NO:1 under moderately stringent conditions, e.g.,hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecylsulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at42° C. (see Ausubel et al editors, Protocols in Molecular Biology, Wileyand Sons, 1994, pp. 6.0.3 to 6.4.10), or conditions which will otherwiseresult in hybridization of sequences that encode a PRRS virus as definedbelow. Modifications in hybridization conditions can be empiricallydetermined or precisely calculated based on the length and percentage ofguanosine/cytosine (GC) base pairing of the probe. The hybridizationconditions can be calculated as described in Sambrook, et al., (Eds.),Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.

In another embodiment, a second nucleotide sequence is homologous to SEQID NO:1 (or any other sequence of the invention) if it hybridizes to thecomplement of SEQ ID NO:1 under highly stringent conditions, e.g.hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at65° C., and washing in 0.1xSSC/0.1% SDS at 68° C., as is known in theart (Ausebel et al. Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York, 1989.

It is furthermore to be understood that the isolated polynucleotidemolecules and the isolated RNA molecules of the present inventioninclude both synthetic molecules and molecules obtained throughrecombinant techniques, such as by in vitro cloning and transcription.

Polynucleotide molecules can be genetically mutated using recombinanttechniques known to those of ordinary skill in the art, including bysite-directed mutagenesis, or by random mutagenesis such as by exposureto chemical mutagens or to radiation, as known in the art.” Themutations may be carried out by standard methods known in the art, e.g.site directed mutagenesis (see e.g. Sambrook et al. (1989) MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) of an infectious copy as described(e.g. Meulenberg et al., Adv. Exp. Med. Biol., 1998, 440:199-206).

Accordingly, the subject invention further provides a method for makinga genetically modified North American PRRS virus, which method comprisesmutating the DNA sequence encoding an infectious RNA molecule whichencodes the PRRS virus as described above, and expressing thegenetically modified PRRS virus using a suitable expression system. Agenetically modified PRRS virus can be expressed from an isolatedpolynucleotide molecule using suitable expression systems generallyknown in the art, examples of which are described in this application.For example, the isolated polynucleotide molecule can be in the form ofa plasmid capable of expressing the encoded virus in a suitable hostcell in vitro, as is described in further detail below.

The North American PRRSV N protein sequences are highly conserved andthe reported sequences have about 93-100% identity with each other. TheNorth American and European PRRSV N proteins are about 57-59% identicaland share common structural motifs. Generally, when comparing PRRSencoding sequences and isolates, which might be numbered differently asto specific nucleotides or encoded amino acids, identification of theproper regions are readily achieved by identifying preservedcharacteristic amino acids in a PRRS strain of interest and aligning itwith a reference strain.

Recombinant DNA technology comprises extremely varied and powerfulmolecular biology techniques aimed at modifying nucleic acids at the DNAlevel and makes it possible to analyze and modify genomes at themolecular level. In this respect, viruses such as the PRRS virus becauseof the modest size of its genome is particularly amenable to suchmanipulations. However, recombinant DNA technology is not immediatelyapplicable to non-retroviral RNA viruses because these viruses do notencompass a DNA intermediate step in their replication. For suchviruses, infectious cDNA clones have to be developed before recombinantDNA technology can be applied to their genome to generate modifiedvirus. Infectious clones can be derived through the construction offull-length (genomic length) cDNA (here used in the broad sense of a DNAcopy of RNA and not only in the strict sense of a DNA copy of mRNA) ofthe virus under study, after which an infectious transcript issynthesized in vivo in cells transfected with the full-length cDNA, butinfectious transcripts can also be obtained by in vitro transcriptionfrom full-length cDNA in a plasmid having a prokaryotic promoter in thepresence of a transcription cocktail, or again in vitro using ligatedpartial-length cDNA fragments that comprise the full viral genome. Inall cases, the transcribed RNA carries all the modifications that havebeen introduced to the cDNA and can be used to further passage the thusmodified virus.

The preparation of an infectious clone of a European PRRS virus isolateor Lelystad virus is described in U.S. Pat. No. 6,268,199 which ishereby fully incorporated by reference. The preparation of an infectiouscDNA clone of a North American PRRS virus isolate designated P129 (Leeet al., 2005; Yoo et al., 2004) is described in U.S. Pat. No. 6,500,662which is hereby incorporated fully by reference. The sequence of P129cDNA is disclosed in Genbank Accession Number AF494042 and in U.S. Pat.No. 6,500,662. Our work below makes use of such an infectious clonewhich in the context of a plasmid is expressed by the CMV immediateearly promoter and has been designated pCMV-S-P129 and is also disclosedwithin U.S. Pat. No. 6,500,662. As described in U.S. Pat. No. 6,500,662there are other plasmids and promoters suitable for use here.

Given the complete sequence of any open reading frame of interest andthe location of an amino acid residue of interest, one of ordinary skillneed merely consult a codon table to design changes at the particularposition desired.

Codons constitute triplet sequences of nucleotides in mRNA and theircorresponding cDNA molecules. Codons are characterized by the baseuracil (U) when present in a mRNA molecule but are characterized by basethymidine (T) when present in DNA. A simple change in a codon for thesame amino acid residue within a polynucleotide will not change thesequence or structure of the encoded polypeptide. It is apparent thatwhen a phrase stating that a particular 3 nucleotide sequence“encode(s)” any particular amino acid, the ordinarily skilled artisanwould recognize that the table above provides a means of identifying theparticular nucleotides at issue. By way of example, if a particularthree nucleotide sequence encodes lysine, the table above discloses thatthe two possible triplet sequences are AAA and AAG. Glycine is encodedby GGA, GGC, GGT (GGU if in RNA) and GGG. To change a lysine to glycineresidue in an encoded protein one might replace a AAA or AAG tripletwith any of by GGA and GGC, GGT or GGG in the encoding nucleic acid.

As aforementioned, the present invention is directed to the provision ofvaccine strains of PRRS wherein host responses to the virus that aremediated by interferon pathways, among other responses, are notdownregulated. As described in detail below, there are variousmodifications to the viral genome that are effective in this regard,particularly those found in ORF1a as disclosed herein, and combinationsthereof. It should be noted that similar modification points can befound in additional open reading frames of the PRRS genome, as alsodisclosed herein (see Table 9).

It is noteworthy that certain other prior approaches to modification ofthe PRRS polynucleotide have been successful in order to attenuate thePRRS virus, possibly providing suitability for vaccine use, although theexact cause of the resultant attenuations is not generally known. Forexample, it has been disclosed to attenuate a virulent PRRS virus bymutating or deleting the NLS-2 region, NoLS region, or the NES region inthe nucleocapsid or N protein (encoded by ORF7) of the virus, to includea deletion in open reading frame 7 (ORF7). In another aspect the ORF7deletion is within the sequence encoding a nuclear localization signal(NLS) of a capsid protein. The ORF7 deletion within the sequenceencoding an NLS may include deletion of one or more amino acids atpositions 43-48 or deletion of an amino acid at either or both positions43 and 44. See, for example, the entire disclosure of U.S. Pat. No.7,544,362 which is incorporated by reference. The nucleocapsid protein(N) of PRRSV, which is encoded by ORF7, is a small basic protein that isphosphorylated (Wootton, Rowland, and Yoo, 2002) and forms homodimers(Wootton and Yoo, 2003). The crystal structure has recently beendetermined (Doan and Dokland, 2003). The N protein appears to havemultiple functions in the infected cell. In addition to forming aspherical capsid structure into which genomic RNA is packaged, a processthat takes place in the cytoplasm, a portion of N protein is transportedinto the nucleus and specifically to the nucleolus of the infected cell.The amino acid sequence of N protein contains two nuclear localizationsignals (NLS), a nucleolar localization signal (NoLS), and a nuclearexport signal (NES) that facilitate transport into the nucleus andnucleolus, and export from the nucleus, respectively (Rowland et al.,1999; Rowland et al., 2003; Rowland and Yoo, 2003). While in thenucleolus, the N protein interacts with the small nucleolarRNA-associated protein fibrillarin and may regulate rRNA processing andribosome biogenesis in the infected cell in order to favor virusreplication (Yoo et al., 2003). Viral mutations of this type arevaluable, either alone or in combination with other attenuatingmutations, for designing novel PRRS vaccines. In another example of aPRRS virus that has been attenuated, modified to ORF1a was employed.Deletion of the DNA sequence encoding the antigenic epitope betweenamino acids 616 to 752 in the hypervariable region in the nonstructuralprotein 2 coding region of ORF1a was employed, see. U.S. Pat. No.7,618,797, which is incorporated by reference in its entirety.

Studies on the immunobiology of PRRS virus are suggestive that theinteraction of PRRS virus with PDCs merits examination. This cell typerepresents 0.2%-0.8% of peripheral blood mononuclear cells in humans,mice, rats, pigs and monkeys. Despite its scarcity, this cell is animportant component of the innate immune system and is capable ofsecreting copious amounts of IFN-α following viral stimulation. It isthrough the secretion of IFN-α that PDCs play a major role in regulatingantiviral innate and adaptive immunity since they promote the functionof natural killer cells, B cells, and T cells. Furthermore, thematuration of porcine monocyte derived dendritic cells (MoDC) is aidedby the IFN-α secreted by PDCs resulting in an enhanced ability of MoDCsto present antigen and activate T cells. At a later stage of viralinfection, PDCs differentiate into a unique type of mature dendriticcell, which directly regulates the function of T cells and direct thedifferentiation of T cells into cells capable of secreting IFN-γ, whichis a major mediator of antiviral immunity against viruses including PRRSvirus. Not surprisingly there are human viruses, such as respiratorysyncitial virus and measles virus, which are known to suppress theability of PDCs to secrete IFN-α. This inhibitory effect is thought toplay a role in the predominance of a humoral immune response and theassociated immunopathology observed as a result of the infection withthese viruses, as well as in the increased susceptibility of the host tosecondary bacterial and viral infections.

In contrast, the wild-type PRRSV isolates as well as both of theIngelvac PRRS vaccine strains (see Examples 5 and following, below)inhibited the ability of purified populations of porcine PDC to produceIFN-α, while the novel P129-PK-FL and P129-PK-d43/44 virus stocks (seebelow) exhibited a minimal to nil inhibitory effect on this PDCfunction. The significance of these observations resides, in part, onthe importance of IFN-α in regulating the development of the adaptiveimmune response to viruses. Accordingly, it is very likely that anattenuated virus vaccine derived from a minimally IFN-α suppressingvirus would elicit a strong antiviral protective immune response. It haspreviously been demonstrated the adjuvant effect of IFN-α on theIngelvac PRRS MLV vaccine induced virus-specific T cell mediated IFN-γresponse, and that the intensity of the virus-specific T cell mediatedIFN-γ response elicited by the vaccine has a positive correlation withprotective immunity against the virus under field and laboratoryconditions. Accordingly, although not being limited as to theory, itwould be reasonable to expect that the cell-mediated immune response andlevel of protective immunity elicited by a non-IFN-α-inhibitory PRRSVwill be significantly greater than that of a PRRSV isolate exhibitingthe wild-type (IFN-α inhibitory) phenotype.

Referring to the present invention, it is notable that the P129-PK-FLvirus as well as all five deletion mutants derived from thepCMV-S-P129-PK infectious cDNA clone lost the ability to inhibit IFN-αproduction. Therefore this unusual phenotype can not be solely due tothe deletions, but must be due at least in part to genetic changes thatbecame fixed during construction of the infectious clone. Interestingly,uncloned P129 virus that was serially passaged 63 times on PK-9 cellsretained the ability to inhibit IFN induction (Table 1). The most likelyexplanation for the common IFN phenotype seen in all infectiousclone-derived viruses is the incorporation of one or more mutationsduring the generation of the infectious clone. These mutations wouldhave existed, possibly at low levels, in the viral RNA used to constructthe infectious clone. Ultimately, the mutations may have existed in theoriginal (passage 0) virus in the pig or they may have been generatedand enriched during the process of adapting the virus to growth on PK-9cells for 16 passages. The possibility that the mutations were theresult PCR-induced errors or cloning artifacts cannot be ruled out. Atany rate, the mutation(s) responsible for the loss of the IFN-αinhibitory function became “fixed” during infectious clone construction,and would be expected to be present in all viruses derived from thisparticular infectious clone.

The possibility that mutations responsible for the altered IFN-αinhibition phenotype pre-existed in the viral RNA used to construct thecDNA clone is likely, given that PRRSV is known to readily generaterandom genetic diversity as a result of errors by the viralRNA-dependent RNA polymerase. Virus quasi-species are comprised of aheterogeneous mixture of closely related genetic variants that naturallyappear during virus replication in vivo. Even more relevant is theobservation of virus quasi-species after multiple in vitro passages ofPRRSV derived from an infectious cDNA clone. This is notable since thestarting population of virus genomes in previously conducted studiesconsisted of a genetically homogenous population, and sequence diversitywas rapidly generated during passage in cell culture. In the currentstudy, the level of genomic heterogeneity would have been higher, sincethe original P129 virus had not been cloned (biologically ormolecularly) prior to the 16 PK-9 passages leading up to construction ofthe infectious clone. Thus the chance selection of a PRRSV RNA variantresponsible for the loss of IFN-α inhibition function from among thequasi-species, and incorporation into the pCMV-S-P129-PK17-FL infectiouscDNA clone seems plausible. The incorporation of these mutations into aninfectious clone might be considered fortuitous, under somecircumstances, given that all derivative viruses should share thisdistinct biological phenotype which may prove important for thedevelopment of effective next-generation PRRS vaccines.

The wild-type PRRS virus strain P129, like other strains of this virus,exhibited a strong inhibitory effect on the ability of peripheral bloodmononuclear cells (PBMCs) and plasmacytoid dendritic cells (PDCs) toproduce interferon (IFN)-α. On the other hand, virus derived from aninfectious cDNA clone of P129 (pCMV-S-P129-PK17-FL) exhibited asignificant reduction in the IFN-α inhibitory phenotype. This infectiousclone was constructed from virus which was previously adapted to grow onthe CD163-expressing porcine kidney cell line PK-9 over the course of 16serial passages (see U.S. Pat. No. 7,754,464 which is incorporated byreference in its entirety). The IFN-α inhibitory phenotype of P129-PK-FLand P129-PK-d43/44 ranged from low to negligible and was in markedcontrast to that exhibited by either of the two Ingelvac PRRS modifiedlive virus vaccine strains, both of which were highly inhibitory. Theseresults indicate that the P129-PK-FL and P129-PK-d43/44 viruses arebiologically distinct from the parental low-passage P129 isolate, otherwild-type PRRS viruses, and both Ingelvac PRRS vaccines. The potentialimplications of the reduced IFN-α-inhibitory phenotype, as well aspossible reasons for the phenotypic change, are discussed.

Amino Acid Modifications of the Invention

According to the practice of the present invention, novel isolates ofPRRS, whether of North American or Chinese genotypes, may befield-identified that contain specific contain amino acids at specificpositions in the proteins encoded from ORF1, and which confer desirablephenotypes on these viruses. In the alternative, as aforementioned,standard genetic procedures may be employed to modify the geneticsequence (and thus the amino acid sequence) of the encoded ORF1 protein,again to produce modified North American and Chinese PRRS viruses, andinfectious clones, and vaccines therefrom, all which provide suchphenotypes. In preferred examples the phenotypes include, withoutlimitation, decreased interferon-α inhibitory effect as compared towild-type PRRS virus, and, optionally, the ability to reproduce orpersist in a host animal (a pig) while triggering a robust immuneresponse, but with little detectable pathology.

Thus, in the practice of the invention, North American PRRS strains orisolates that may serve as useful starting points include thosedisclosed, for example in U.S. Pat. Nos. 6,500,662; 7,618,797;7,691,389, 7,132,106; 6,773, 908; 7,264,957; 5,695,766; 5,476,778;5,846,805; 6,042,830; 6,982,160; 6,241,990; and 6,110,468. In regard ofChinese PRRS strains and isolates that may serve as useful startingpoints, see for example, published Chinese application CN200910091233.6from Chinese application CN201633909 pertaining to the TJM-92 virus.

In connection with the discussion that follows, internationallyrecognized single and three-letter designations are used for the mostcommon amino acids encoded by DNA: alanine (Ala, A); arginine (Arg, R);asparagine (Asn, N); aspartic acid (Asp, D); cysteine (Cys, C); glutamicacid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); histidine (H is,H); isoleucine (Ile, I); leucine (Leu, L); lysine (Lys, K); methionine(Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (ser, S);threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y) and Valine(Val, V).

Tables 9 and 10 identify observed amino acid changes responsible forattenuation of virulence in North American (and Chinese) PRRS whichcorrelate with reduced inhibition of interferon alpha activity, therebypermitting a safe and robust immune response to vaccines. Table 10identifies highly preferred amino acid modifications in this regard,within ORF1a, and shows how these mutations have arisen in regard ofpassaging from other P129 cultures (it should be noted that theinspection of this history also facilitates design of mutagenesisstrategies to (re) construct encoding DNA having any of the amino acidchanges of the invention, as needed). In this regard, the most preferredamino acid improvements to ORF1a (as evidenced by P129 passage 52include: asparagine at 182, asparagine at 189, tyrosine at 273,histidine at 302, threonine at 665, cysteine at 943, threonine at 1429,alanine at 1505, asparagine at 2410, which also potentially addsnumerous glycoslation opportunities and which may further alter proteinfunction.

Accordingly, the invention provides an isolated Porcine Reproductive andRespiratory Syndrome Virus (PRRS) wherein the protein thereof encoded byORF1a is selected from a group consisting of those amino acid sequencesthat contain any of:

an amino acid N within the amino acid sequence AMANVYD (SEQ ID NO: 9);an amino acid N within the amino acid sequence IGHNAVM (SEQ ID NO: 12);an amino acid D within the amino acid sequence TVPDGNC (SEQ ID NO: 15);an amino acid Y within the amino acid sequence CWWYLFD (SEQ ID NO: 18);an amino acid H within the amino acid sequence HGVHGKY (SEQ ID NO: 21);an amino acid V within the amino acid sequence AAKVDQY (SEQ ID NO: 24);an amino acid T within the amino acid sequence PSATDTS (SEQ ID NO: 27);an amino acid L within the amino acid sequence LNSLLSK (SEQ ID NO: 30).an amino acid C within the amino acid sequence APMCQDE (SEQ ID NO: 33);an amino acid T within the amino acid sequence CAPTGMD (SEQ ID NO: 36);an amino acid A within the amino acid sequence PKVAKVS (SEQ ID NO: 39);an amino acid I within the amino acid sequence AGEIVGV (SEQ ID NO: 42);an amino acid N within the amino acid sequence ADFNPEK (SEQ ID NO: 45);andan amino acid I within the amino acid sequence QTPILGR (SEQ ID NO: 48).

More specifically, the invention provides an isolated PorcineReproductive and Respiratory Syndrome Virus (PRRS) wherein the proteinthereof encoded by ORF1a is selected from a group consisting of thoseamino acid sequences that contain any of:

an amino acid N within the amino acid sequence ANV (see SEQ ID NO: 9);an amino acid N within the amino acid sequence HNA (see SEQ ID NO: 12);an amino acid D within the amino acid sequence PDG (see SEQ ID NO: 15);an amino acid Y within the amino acid sequence WYL (see SEQ ID NO: 18);an amino acid H within the amino acid sequence VHG (see SEQ ID NO: 21);an amino acid V within the amino acid sequence KVD (see SEQ ID NO: 24);an amino acid T within the amino acid sequence ATD (see SEQ ID NO: 27);an amino acid L within the amino acid sequence SLL (see SEQ ID NO: 30).an amino acid C within the amino acid sequence MCQ (see SEQ ID NO: 33);an amino acid T within the amino acid sequence PTG (see SEQ ID NO: 36);an amino acid A within the amino acid sequence VAK (see SEQ ID NO: 39);an amino acid I within the amino acid sequence EIV (see SEQ ID NO: 42);an amino acid N within the amino acid sequence FNP (see SEQ ID NO: 45);andamino acid I within the amino acid sequence PIL (see SEQ ID NO: 48).

As aforementioned, there are numerous known strains and isolates ofNorth American and Chinese PRRS, and novel strains continue to evolve orto be isolated. Although a high level of amino acid sequence homologyexists between all these strains, those skilled in the art willimmediately recognize that some variation does exist, and indeedadvantage can be taken of these differences and similarities to furtherimprove the phenotypic properties of all vaccine strains.

First, in regard of all of the amino acid motifs defined by SEQ ID NOSas specified directly (on Pages 27-28) above, the underlined andpreferred amino acids (as provided from P129 passage 52) generallyremain fully beneficial even if adjacent amino acids have otherwisechanged from the specified SEQ ID NO sequences. Thus in regard ofAMANVYD (SEQ ID NO: 9), as a specific and representative example, it isgenerally possible to inspect the corresponding ORF1-expressed proteinsequence from any North American or Chinese PRRS, to find thecorresponding amino acid motif, even if additional changes have occurredin such other strains, as a result of evolution, causing substitutionsand/or deletions or additions. As will be appreciated by those skilledin the art, the preferred amino acid changes evidenced by P129 passage52 should thus also remain operable in spite of other changes in overallamino acid sequence that are directly 5′ or 3′ to the specified aminoacid of Passage 52. This will be so especially if the comparative aminoacid changes are considered conservative. Thus in regard of AMANVYD (SEQID NO: 9), and the subsequence ANV thereof, it should be readilypossible to identify the comparable motif in another PRRS strain if, forexample, the valine therein is replaced by isoleucine or leucine, or anyother residue, or if a residue is simply missing or an additionalresidue added. Numerous computer programs exist to identify alignmentsand thus determine if polypeptide sequence motifs correspond, forexample the so-called Blosum tables (based on a given level of percentidentity), see S. Henikoff et al. “Amino Acid Substitution matrices fromprotein blocks”, Proc Natl Acad Sci, USA, 89(22), pp. 10915-10919, Nov.15, 1992., and see also A. L. Lehninger et al. Principles ofBiochemistry, 2005, MacMillan and Company, 4^(th) edition. Conservativeamino acid changes are also recognized based on categorization into 5overall groups: sulfydryl (Cys); aromatic (Phe, Tyr, and Trp); basic(Lys, Arg, His); aliphatic (Val, Ileu, Leu, Met), and hydrophilic (Ala,Pro, Gly Glu, Asp, Gin, Asn, Ser and Thr). Thus it is within thepractice of the invention to modify any North American or Chinese PRRSencoding nucleotide sequence to incorporate at the appropriate andcorresponding position, any of the amino acid changes specified for P129passage 52, even if one or more of the other amino acids adjacent to thedesignated position have been added, deleted or substituted.

Additionally, based on similar principles, those skilled in the art willrecognize that once a preferred amino acid is identified from thespecific Passage 52 changes identified for ORF1a according to thepractice of the present invention, that conservative replacements forany such passage 52 amino acids can then also be used, either in P129variants, or in regard of any other North American or Chinese strains,with substantial preservation of the intended passage 52 phenotype.Thus, as representative examples: in regard of SLL (within SEQ ID NO:30), the designated leucine residue may be further replaced withisoleucine, valine or methionine; in regard of FNP (within SEQ ID NO:45), the designated asparagine may be replaced with any of Ala, Pro,Gly, Glu, Asp, Gin, Ser and Thr; and in regard of VAK (see SEQ ID NO:39), the designated alanine may be replaced with any of Asn, Pro, Gly,Glu, Asp, Gin, Ser and Thr; all and the like, although it will bereadily recognized that it is not a requirement of the present inventionthat any such replacement amino acids work as well as originallyidentified unique Passage 52 amino acid changes, at the specifiedlocations. Of course, use of standard conservative amino acid changesaccording to any other recognized model also is practiced in the presentinvention. For example, and including vice-versa in all cases, Asp forGlu and vice versa, Asn for Gin, Arg for Lys, Ser for Cys or Thr, Phefor Tyr, Val for Leu or Ileu, Ala for Gly, and the like.

Further, within the practice of the present invention, although any ofthe individual Passage 52 amino acid changes (as identified for ORF1aabove) can be usefully placed in any North American of Chinese PRRS withdesired phenotypic effect, it is further preferred to in include as manyof the Table 9 or Table 10 amino acid selections as possible in a finalconstruct, as typically provided for by appropriate modification of theencoding polynucleotide sequence. Thus, the practice of the presentinvention includes the provision of Chinese or North American PRRSviruses (and corresponding encoding polynucleotides) that provide for 2,3, 4, 5, 6, 7, 8, 9, and up to any of the approximately 17 identifiedPassage 52 ORF1a changes, (Table 9) all within a final viral sequence,to include any specific pairs, triplets, or other higher combinations ofall the total identified Passage 52 amino acid changes. Such amino acidchanges may, of course, be introduced into the corresponding encodingnucleotide sequences of the virus by site directed mutagenesis, PCR, andother techniques as are well known in the art.

To demonstrate that a particular genetically modified strain isattenuated an experiment described as follows may be used.

At least 10 gilts per group are included in each trial, which arederived from a PRRSV-free farm. Animals are tested free of PRRS virusspecific serum antibodies and negative for PRRSV. All animals includedin the trial are of the same source and breed. The allocation of theanimals to the groups is randomized.

Challenge is performed at day 90 of pregnancy with intranasalapplication of 1 ml PRRSV with 10⁵ TCID₅₀ per nostril. There are atleast three groups for each test setup: One group for P129 challenge;one test group for challenge with the possibly attenuated virus; and onestrict control group.

The study is deemed valid when the strict controls stay PRRSV-negativeover the time course of the study and at least 25% less live healthypiglets are born in the P129 challenged group compared to the strictcontrols.

Attenuation, in other words less virulence, is defined as thestatistical significant change of one or more parameters determiningreproductive performance or other symptomology:

Significant reduction in at least one of the following parameters forthe test group (possibly attenuated virus) compared to the unmodifiedparental strain infected group would be an indication of attenuation:a) frequency of stillbornsb) abortion at or before day 112 of pregnancyc) number of mummified pigletsd) number of less lively and weak pigletse) pre-weaning mortalityFurthermore a significant increase in one of the following parametersfor the test group compared the unmodified parental strain infectedgroup is preferred:f) number of piglets weaned per sowg) number of live healthy piglets born per sow

In the alternative, respiratory symptoms and other symptoms of PRRSVinfection could be examined to establish attenuation.

An attenuated strain is valuable for the formulation of vaccines. Thepresent vaccine is effective if it protects a pig against infection by aPRRS virus. A vaccine protects a pig against infection by a PRRS virusif, after administration of the vaccine to one or more unaffected pigs,a subsequent challenge with a biologically pure virus isolate (e.g., VR2385, VR 2386, P129 etc.) results in a lessened severity of any gross orhistopathological changes (e.g., lesions in the lung) and/or of symptomsof the disease, as compared to those changes or symptoms typicallycaused by the isolate in similar pigs which are unprotected (i.e.,relative to an appropriate control). More particularly, the presentvaccine may be shown to be effective by administering the vaccine to oneor more suitable pigs in need thereof, then after an appropriate lengthof time (e.g., 4 weeks), challenging with a large sample (10⁽³⁻⁷⁾TCID₍₅₀₎) of a biologically pure PRRSV isolate. A blood sample is thendrawn from the challenged pig after about one week, and an attempt toisolate the virus from the blood sample is then performed. Isolation ofa large amount of the virus is an indication that the vaccine may not beeffective, while isolation of reduced amounts of the virus (or no virus)is an indication that the vaccine may be effective.

Thus, the effectiveness of the present vaccine may be evaluatedquantitatively (i.e., a decrease in the percentage of consolidated lungtissue as compared to an appropriate control group) or qualitatively(e.g., isolation of PRRSV from blood, detection of PRRSV antigen in alung, tonsil or lymph node tissue sample by an immunoassay). Thesymptoms of the porcine reproductive and respiratory disease may beevaluated quantitatively (e.g., temperature/fever) orsemi-quantitatively (e.g., the presence or absence of one or moresymptoms or a reduction in severity of one or more symptoms, such ascyanosis, pneumonia, lung lesions etc.).

An unaffected pig is a pig which has either not been exposed to aporcine reproductive and respiratory disease infectious agent, or whichhas been exposed to a porcine reproductive and respiratory diseaseinfectious agent but is not showing symptoms of the disease. An affectedpig is one which shows symptoms of PRRS or from which PRRSV can beisolated.

Vaccines of the present invention can be formulated following acceptedconvention to include acceptable carriers for animals, including humans(if applicable), such as standard buffers, stabilizers, diluents,preservatives, and/or solubilizers, and can also be formulated tofacilitate sustained release. Diluents include water, saline, dextrose,ethanol, glycerol, and the like. Additives for isotonicity includesodium chloride, dextrose, mannitol, sorbitol, and lactose, amongothers. Stabilizers include albumin, among others. Other suitablevaccine vehicles and additives, including those that are particularlyuseful in formulating modified live vaccines, are known or will beapparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Science, 18th ed., 1990, Mack Publishing, which isincorporated herein by reference.

Vaccines of the present invention can further comprise one or moreadditional immunomodulatory components such as, e.g., an adjuvant orcytokine, among others. Non-limiting examples of adjuvants that can beused in the vaccine of the present invention include the RIBI adjuvantsystem (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminumhydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as,e.g., Freund's complete and incomplete adjuvants, Block copolymer(CytRx, Atlanta, Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.),SAF-M (Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil Aor other saponin fraction, monophosphoryl lipid A, and Avridinelipid-amine adjuvant. Non-limiting examples of oil-in-water emulsionsuseful in the vaccine of the invention include modified SEAM62 and SEAM1/2 formulations. Modified SEAM62 is an oil-in-water emulsion containing5% (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICISurfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5%(v/v) ethanol, 200 pg/ml Quil A, 100 [mgr]g/ml cholesterol, and 0.5%(v/v) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising5% (v/v) squalene, 1% (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween 80detergent, 2.5% (v/v) ethanol, 100 .mu.g/ml Quil A, and 50 .mu.g/mlcholesterol. Other immunomodulatory agents that can be included in thevaccine include, e.g., one or more interleukins, interferons, or otherknown cytokines.

Vaccines of the present invention can optionally be formulated forsustained release of the virus, infectious RNA molecule, plasmid, orviral vector of the present invention. Examples of such sustainedrelease formulations include virus, infectious RNA molecule, plasmid, orviral vector in combination with composites of biocompatible polymers,such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid),methylcellulose, hyaluronic acid, collagen and the like. The structure,selection and use of degradable polymers in drug delivery vehicles havebeen reviewed in several publications, including A. Domb et al., 1992,Polymers for Advanced Technologies 3: 279-292, which is incorporatedherein by reference. Additional guidance in selecting and using polymersin pharmaceutical formulations can be found in texts known in the art,for example M. Chasin and R. Langer (eds), 1990, “Biodegradable Polymersas Drug Delivery Systems” in: Drugs and the Pharmaceutical Sciences,Vol. 45, M. Dekker, N.Y., which is also incorporated herein byreference. Alternatively, or additionally, the virus, plasmid, or viralvector can be microencapsulated to improve administration and efficacy.Methods for microencapsulating antigens are well-known in the art, andinclude techniques described, e.g., in U.S. Pat. No. 3,137,631; U.S.Pat. No. 3,959,457; U.S. Pat. No. 4,205,060; U.S. Pat. No. 4,606,940;U.S. Pat. No. 4,744,933; U.S. Pat. No. 5,132,117; and InternationalPatent Publication WO 95/28227, all of which are incorporated herein byreference.

Liposomes can also be used to provide for the sustained release ofvirus, plasmid, or viral vector. Details concerning how to make and useliposomal formulations can be found in, among other places, U.S. Pat.No. 4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,921,706; U.S.Pat. No. 4,927,637; U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050;and U.S. Pat. No. 5,009,956, all of which are incorporated herein byreference.

An effective amount of any of the above-described vaccines can bedetermined by conventional means, starting with a low dose of virus,viral protein plasmid or viral vector, and then increasing the dosagewhile monitoring the effects. An effective amount may be obtained aftera single administration of a vaccine or after multiple administrationsof a vaccine. Known factors can be taken into consideration whendetermining an optimal dose per animal. These include the species, size,age and general condition of the animal, the presence of other drugs inthe animal, and the like. The actual dosage is preferably chosen afterconsideration of the results from other animal studies (see, forexample, Examples 2 and 3 below).

One method of detecting whether an adequate immune response has beenachieved is to determine seroconversion and antibody titer in the animalafter vaccination. The timing of vaccination and the number of boosters,if any, will preferably be determined by a doctor or veterinarian basedon analysis of all relevant factors, some of which are described above.

The effective dose amount of virus, protein, infectious DNA molecule,plasmid, or viral vector, of the present invention can be determinedusing known techniques, taking into account factors that can bedetermined by one of ordinary skill in the art such as the weight of theanimal to be vaccinated. The dose amount of virus of the presentinvention in a vaccine of the present invention preferably ranges fromabout 10¹ to about 10⁹ pfu (plaque forming units), more preferably fromabout 10² to about 10⁸ pfu, and most preferably from about 10³ to about10⁷ pfu. The dose amount of a plasmid of the present invention in avaccine of the present invention preferably ranges from about 0.1 mg toabout 100 mg, more preferably from about 1 mg to about 10 mg, even morepreferably from about 10 mg to about 1 mg. The dose amount of aninfectious DNA molecule of the present invention in a vaccine of thepresent invention preferably ranges from about 0.1 mg to about 100 mg,more preferably from about 1 mg to about 10 mg, even more preferablyfrom about 10 mg to about 1 mg. The dose amount of a viral vector of thepresent invention in a vaccine of the present invention preferablyranges from about 10¹ pfu to about 10⁹ pfu, more preferably from about10² pfu to about 10⁸ pfu, and even more preferably from about 10³ toabout 10⁷ pfu. A suitable dosage size ranges from about 0.5 ml to about10 ml, and more preferably from about 1 ml to about 5 ml.

Suitable doses for viral protein or peptide vaccines according to thepractice of the present invention range generally from 1 to 50micrograms per dose, or higher amounts as may be determined by standardmethods, with the amount of adjuvant to be determined by recognizedmethods in regard of each such substance. In a preferred example of theinvention relating to vaccination of swine, an optimum age target forthe animals is between about 1 and 21 days, which at pre-weening, mayalso correspond with other scheduled vaccinations such as againstMycoplasma hyopneumoniae or PCV. Additionally, a preferred schedule ofvaccination for breeding sows would include similar doses, with anannual revaccination schedule.

An effective amount of any of the above-described vaccines can bedetermined by conventional means, starting with a low dose of virus,plasmid or viral vector, and then increasing the dosage while monitoringthe effects. An effective amount may be obtained after a singleadministration of a vaccine or after multiple administrations of avaccine. Known factors can be taken into consideration when determiningan optimal dose per animal. These include the species, size, age andgeneral condition of the animal, the presence of other drugs in theanimal, and the like. The actual dosage is preferably chosen afterconsideration of the results from other animal studies.

One method of detecting whether an adequate immune response has beenachieved is to determine seroconversion and antibody titer in the animalafter vaccination. The timing of vaccination and the number of boosters,if any, will preferably be determined by a doctor or veterinarian basedon analysis of all relevant factors, some of which are described above.

The effective dose amount of virus, infectious RNA molecule, plasmid, orviral vector, of the present invention can be determined using knowntechniques, taking into account factors that can be determined by one ofordinary skill in the art such as the weight of the animal to bevaccinated. By way of example, vaccines may be delivered orally,parenterally, intradermally, subcutaneously, intramuscularly,intranasally or intravenously. Oral delivery may encompass, for example,adding the compositions to the feed or drink of the animals. Factorsbearing on the vaccine dosage include, for example, the weight and ageof the pig.

The present invention further provides a method of preparing a vaccinecomprising a PRRS virus, infectious RNA molecule, plasmid, or viralvector described herein, which method comprises combining an effectiveamount of one of the PRRS virus, infectious RNA molecule, plasmid, orviral vector of the present invention, with a carrier acceptable forpharmaceutical or veterinary use.

In addition the live attenuated vaccine of the present invention can bemodified as described in U.S. Pat. No. 6,500,662 to encode aheterologous antigenic epitope which is inserted into the PRRS viralgenome using known recombinant techniques. See also U.S. Pat. No.7,132,106 which is incorporated by reference in its entirety. Antigenicepitopes useful as heterologous antigenic epitopes for the presentinvention include antigenic epitopes from a swine pathogen other thanPRRS virus which include, but are not limited to, an antigenic epitopefrom a swine pathogen selected from the group consisting of porcineparvovirus, porcine circovirus, a porcine rotavirus, swine influenza,pseudorabies virus, transmissible gastroenteritis virus, porcinerespiratory coronavirus, classical swine fever virus, African swinefever virus, encephalomyocarditis virus, porcine paramyxovirus, torqueteno virus, Actinobacillus pleuropneumoniae, Actinobacillus suis,Bacillus anthraci, Bordetella bronchiseptica, Clostridium haemolyticum,Clostridium perfringens, Clostridium tetani, Escherichia coli,Erysipelothrix rhusiopathiae, Haemophilus parasuis, Leptospira spp.,Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynovia,Pasteurella multocida, Salmonella choleraesuis, Salmonella typhimurium,Streptococcus equismilis, and Streptococcus suis. Nucleotide sequencesencoding antigenic epitopes from the aforementioned swine pathogens areknown in the art and can be obtained from public gene databases on theworldwide web, such as at Genbank from the (USA) National Center forBiotechnology Information.

Additional features and variations of the invention will be apparent tothose skilled in the art from the entirety of this application,including the detailed description, and all such features are intendedas aspects of the invention. Likewise, features of the inventiondescribed herein can be re-combined into additional embodiments thatalso are intended as aspects of the invention, irrespective of whetherthe combination of features is specifically mentioned above as an aspector embodiment of the invention. Also, only such limitations which aredescribed herein as critical to the invention should be viewed as such;variations of the invention lacking limitations which have not beendescribed herein as critical are intended as aspects of the invention.It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the invention.

The following examples are intended to illustrate but not limit theinvention.

Example 1 Adaptation and Attenuation of Prrsv Isolate P129 to Pk-9 Cells

Virulent PRRS isolate P129 was isolated from a sick pig in Indiana in1995 at the Animal Disease Diagnostic Laboratory of Purdue University. Aserum sample from this pig was passaged once in a high health status pigto expand serum and lung homogenate stocks. Viral RNA was extracted fromthe serum and lung homogenate and used to determine the complete genomeconsensus sequence of P129 passage 0 virus. RNA was first primed withrandom hexamers and used to synthesize cDNA. The genome was amplified inthree overlapping pieces using high fidelity (proofreading) PCR. The PCRproducts from three separate PCR reactions (per genome segment) were T/Acloned and used for DNA sequencing to generate a full length genomeconsensus sequence (see SEQ ID NO:1).

An aliquot of the same pig serum used for DNA sequencing, containingP129 passage 0, was used to infect primary porcine alveolar macrophage(PAM) cells. The progeny virus from the PAM infection (passage 1) wasfiltered through a 0.1 micrometer syringe filter and used to infect PK-9cells.

PK-9 cells are a transgenic cell line derived by stably transfecting thePK0809 porcine kidney cell line with a plasmid encoding a deletedversion of the porcine CD163 gene and the neomycin resistance gene.Details of the construction and characterization of the PK-9 cell linehave been described previously.

Adaptation of the passage 1 virus from PAM cells to growth on PK-9 cellswas difficult, and required several attempts with multiple parallellineages. Infection was monitored by immunofluorescence of duplicatewells using FITC-conjugated monoclonal antibody SDOW17 specific for theviral nucleocapsid protein (Rural Technologies Inc, Brookings S. Dak.).Early passages resulted in a few small foci, but did not generate enoughcell-free virus particles to initiate infection of a fresh monolayer.These passages were accomplished by treating the infected monolayer withAccutase (a trypsin substitute) and reseeding the cells in multiplewells with fresh medium, with or without the addition of non-infectedPK-9 cells. After several such passages, some lineages showed a clearincrease in the frequency and size of fluorescent foci. Some of thesehad acquired the ability to be passaged using cell-free virus fluids. Bypassage 17 (1 on PAM cells, 16 on PK-9), one lineage could reliably besustained using dilutions of the cell-free fluids from the previouspassages, and resulted in the infection of the entire monolayer within afew days. The virus did not cause cytopathic effect on PK-9 cells at anypassage level. RNA was extracted from infected PK-9 cells at viruspassage 17 and used to construct an infectious cDNA clone.

Example 2 Construction of an Infectious cDNA Clone of P129-PK Passage 17

An infectious cDNA clone of the P129-PK passage 17 virus wasconstructed, using a backbone plasmid as previously described. Thegenome of the virus was amplified by reverse transcription and PCR inthree overlapping segments, with naturally occurring unique restrictionendonuclease sites in the regions of overlap. The products from threeseparate PCR reactions were cloned and sequenced, and aligned togenerate a consensus sequence for each genome segment. If none of thethree cloned products of a given segment matched the predicted aminoacid sequence of the consensus for that segment, one of the clones wasmodified by subcloning and/or site-directed mutagenesis until it matchedthe predicted amino acid sequence of the consensus. The three genomesegments and the plasmid backbone were joined using standard cloningtechniques and restriction endonuclease sites. The resulting full-lengthclone, designated pCMV-S-P129-PK17-FL, was infectious when transfectedinto PK-9 cells. The sequence of this infectious cDNA clone is given inSEQ ID NO:2. The genome is essentially identical to the passage 17 virusfrom which it was constructed, with authentic termini, and lacks anyinsertions or deletions relative to the consensus sequences of passage17. There are no engineered restriction sites or other targeted changeswithin the viral genome of this infectious cDNA clone.

Nucleotide and amino acid differences between the complete genomeconsensus sequences of P129 passage 0 and the genome sequence ofinfectious clone pCMV-S-P129-PK17-FL are listed individually in Table 6.Table 6 includes all nucleotide differences and resulting amino aciddifferences by genome position. A subset of these mutations areresponsible for the change in phenotype from IFN inhibitory (passage 0)to IFN non-inhibitory (all viruses derived from the passage 17infectious cDNA clone). Table 7 summarizes nucleotide, amino acid, andnon-conserved amino acid differences by PRRSV open reading frame (ORF)or non-1255 structural protein (nsp). For the purposes of Table 7, thefollowing groups of amino acids are among those considered conserved:[K,R], [D,E], [L, I, V, A], and [S,T].

Example 3 Deletion Mutants in P129-PK Passage 17

Deletions in two areas of the genome were engineered into infectiouscDNA clone pCMV-S-P129-PK17-FL, to generate five genetically modifiedinfectious clones.

One area of the genome to undergo modification was the nuclearlocalization sequence (NLS) located at amino acid positions 41-47 of thenucleocapsid protein (encoded by PRRSV ORF 7). Two types of deletionswere made. These deletions have been described previously within thecontext of another PRRSV infectious clone. The wild type sequence ofamino acid residues 41-49 is PG . . . KN. In mutant “d43/44”, also knownas “PG-KS”, lysine residues 43 and 44 are deleted and asparagine residue49 is changed to a serine. In mutant “d43/44/46”, also known as“PG-S-KS”, lysine residues 43, 44, and 46 are deleted and asparagineresidue 49 is changed to a serine. The infectious clones derived frompCMV-S-P129-PK17-FL that incorporate these deletions arepCMV-S-P129-PK17-d43/44 and pCMV-S-P129-PK17-d43/44/46 respectively. SeeU.S. Pat. No. 7,544,362.

The second area of the genome to undergo modification was in thehypervariable region of nsp2, within ORF1a. A deletion of 131 aminoacids (393 nucleotides) has been described previously within the contextof another PRRSV infectious clone. The infectious clone derived frompCMV-S-P129-PK17-FL that incorporate this deletion ispCMV-S-P129-PK17-nsp2.

Infectious clones that combine the NLS and nsp2 deletions were alsogenerated within the pCMV-S-P129-PK17-FL backbone, and these weredesignated pCMV-S-P129-PK17-nsp2-d43/44 andpCMV-S-P129-PK17-nsp2-d43/44/46.

Example 4 Generation and Growth of Viruses on PK-9 Cells

The six infectious clones described in Example 3 were transfected intoPK-9 cells to generate the six viruses as shown in Table 1. Virus wasgenerated from these infectious clones by direct transfection of thecircular plasmid into PK-9 cells using Lipofectamine 2000. Followingtransfection, recovered viruses were again serially passaged on PK-9cells in order to further increase titers and attenuate virulence.Stocks were made for in vitro testing and in vivo evaluation as vaccinecandidates. In the case of P129-PK17-FL virus derived from thenon-modified pCMV-S-P129-PK17-FL infectious clone, the virus wascultured until reaching a total of 52 passages from the pig. Thecomplete genome of this virus was sequenced at passages 24 (SEQ ID NO:3) and 52 (SEQ ID NO:6).

Example 5 Viruses Derived from the P129-PK Passage 17 Infectious cDNAClone have Reduced Ability to Inhibit Ifn-Alpha Induction

Viruses and cells. MARC-145 and ST cells were grown in modified Eagle'smedium (MEM) supplemented with 5% fetal bovine serum (FBS) andantibiotics (50 μg/ml gentamicin, 100 UI penicillin and 100 μg/mlstreptomycin). The porcine alveolar macrophage ZMAC-1 cells were grownin RPMI-1640 supplemented with 10% FBS. TGE virus strain Purdue wasprepared by infection of confluent ST cell monolayers at a multiplicityof 0.01 in modified Eagle's medium. The virus inoculum was removed after1 h and cells were incubated in MEM supplemented with 2.5% FBS at 37° C.in a 5% CO₂ atmosphere. Virus was released by freezing and thawing thecell monolayers after 80% cytopathic effect was observed. The TGE viralstock was centrifuged at 3,500 rpm for 15 min at 4° C. and stored at−80° C. until use. Virus stocks (from PK-9 cells) were as follows:P129-PK-FL and P129-PK-dnsp2-1315 d43/44/46 were at passage 8/25 (8 fromthe infectious clone, 25 from the pig). The other four viruses(P129-PK-d43/44, P129-PK-d43/44/46, P129-PK-dnsp2, andP129-PK-dnsp2-d43/44) were at passage 21/38. Working stocks of variousPRRS viruses were prepared by making a single passage on ZMAC-1 cells,except that commercial vaccines Ingelvac PRRS MLV and Ingelvac PRRS ATPwere reconstituted according to the manufacturer's instructions and useddirectly for infection.

Isolation of porcine PBMC. Fresh heparinized venous blood was dilutedwith Hank's and PBMC were isolated by density centrifugation throughFicoll-Hypaque (Sigma) gradient. After being washed twice in Hank's, thecells were suspended in RPMI medium with L-glutamine (Mediatech)supplemented with 5% fetal bovine serum (Gibco), 100 U/ml penicillin,0.1 mg/ml streptomycin, 1 mM sodium pyruvate, 1× nonessential aminoacids (Mediatech) 100 U/ml gentamicin and 250 mM 2-mercaptoethanol(Sigma).

Purification of porcine plasmacytoid dendritic cells. The purificationof porcine plasmacytoid dendritic cells was done as previously described(Calzada-Nova, submitted), and was based on the characteristicexpression of CD4 and CD172 by these cells (Summerfield et al., 2003)Briefly, fresh porcine PBMC were suspended in PBS with 0.5% BSA andlabeled with optimal amounts of mAb recognizing porcine CD172 (74-22-15,VMRD). Following one wash, the cells were then incubated with secondarygoat anti-mouse antibody conjugated to PE (Southern Biotech) and afterwashing, with FITC labeled anti-CD4 (74-12-4, VMRD). PDCs were sorted ona Reflection Cell Sorter (iCyt), sort gates were set on theCD4⁺/CD172^(low) population. After the sort the purity of the cells wasconfirmed by reanalysis. In all cases, the purity was >95%.

Assay for measurement cytokine secretion. PBMC or PDC were stimulatedfor 16 h (37° C., 5% CO₂) with the different stimulants or weremock-stimulated. After incubation, medium overlaying the stimulatedcells was assayed for the presence of IFN-α using a sandwich ELISAprepared with monoclonal antibodies available commercially (anti-pigIFN-α mAbs K9 and F17). Briefly, Immulon II plates (Dynatech Inc.) werecoated with anti-porcine IFN-α mAb F17 (PBL Laboratories) by overnightincubation at 4° C. followed by blocking with RPMI medium supplementedwith 5% fetal bovine serum. After 1 hour the medium was discarded andfifty microliters of the supernatant to be tested added to the assaywells in duplicate. After a 1 h incubation the assay wells were washed 4times and then incubated sequentially with biotin-labeled, anti-porcineIFN-α mAb K9 (PBL Laboratories), HRP-conjugated streptavidin (ZymedLaboratories), and TMB substrate (KPL). The optical density wasdetermined with an ELISA plate reader.

Viruses derived from the P129-PK passage 17 infectious cDNA clone lackthe ability to inhibit IFN-alpha induction. Working virus stocks wereprepared from of a group of four different PRRS wild-type virus isolates(P3412, P129, IND5, NADC20) utilizing the porcine alveolar macrophagecell line ZMAC-1. Additional stocks were also prepared from derivativesof the first two wild-type viruses which had been adapted to grow incell culture by repeated passage in PK-9, FK.D4 or MARC-145 cells. Threeof the four wild-type isolates (P129, IND5, NADC20) grew readily andefficiently in the ZMAC-1 cells to titers of about 10⁷ TCID₅₀/ml, whilethe P3412 wild-type isolate reached a titer of only 10⁵ TCID₅₀/ml.Notably, stocks of P129 viruses prepared in the ZMAC-1 cell line reached10-fold higher titers than those obtained in the PK-9 or MARC-145 cellsto which the viruses were adapted. Examination of the ability of theseviruses to stimulate IFN-α secretion by PBMC revealed that with oneexception (isolate P3412 clone C), a very small amount (<50 pg) of IFN-αwas secreted by these cells in response to their exposure to any of thePRRS virus stocks tested, which is negligible by comparison to theabundant secretion of IFN-α (17,540 pg) produced by the same cells as aresult of their exposure to the porcine coronavirus, transmissiblegastroenteritis virus (TGEv).

PRRSV is not only unable to stimulate IFN-α production by porcine PBMCbut actively inhibits its production. The inhibitory effects of thePRRSV stocks were determined by measuring the amount of IFN-α secretedby PBMC in response to exposure to TGEv in the presence or absence ofPRRSV. As shown in Table 2, all 4 of the wild-type PRRS virus isolatestested, as well as all of the cell culture adapted derivatives,exhibited a strong inhibitory effect (>80%) on the IFN-α response ofPBMC to TGEv. The analysis of a group of virus stocks derived from aninfectious cDNA clone (pCMV-S-P129-PK17-FL), including full-lengthP129-PK-FL virus and several genetically engineered deletion mutants wasconducted. As shown in Table 3, when compared to the strong inhibitoryeffect (95%) of the parental wild-type isolate P129 (passage 1), theP129-PK-FL virus and all deletion mutants exhibited a significantlyreduced ability to inhibit the induction of IFN- by TGEv in PBMC. Tofurther evaluate the IFN-α phenotype of these viruses, subsequentexperiments were focused on performing direct comparisons between theP129-PK-FL and P129-PK-d43/44 viruses, the parental P129 wild-typestrain, and/or two commercially available modified live PRRSV vaccinesproduced by Boehringer Ingelheim (Ingelvac PRRS MLV and Ingelvac PRRSATP). An additional low-passage reference isolate, NVSL-14, was alsotested. As shown in Table 4, in four independent experiments, P129-PK-FLand P129-PK-d43/44 exhibited significantly lesser IFN-α inhibitoryeffect than the parental P129 virus, the two Ingelvac attenuatedstrains, or the reference strain. In one instance, co-infection with theP129-PK-FL or P129-PK-d43/44 viruses resulted in an apparent enhancementof the IFN-α response to TGEv.

The results shown in Table 2 are indicative of the interferon-αinhibitory effect of wild-type PRRS virus and derivatives adapted togrowth in cell culture. The indicated PRRS virus stocks, were grown inZMAC-1 cells and the titer of these newly generated stocks determinedusing ZMAC-1 cells. The amount of IFN-α present in culture supernatantsof porcine peripheral blood mononuclear cells exposed for 18 h to theindicated PRRS virus stock in the presence or absence of TGE virus wasdetermined by ELISA. *Response to TGEv alone.

The results shown in Table 3 demonstrate the interferon-α inhibitoryeffect of wild-type PRRS virus P129 and its genetically engineeredderivatives adapted to grow in CD163-expressing PK-9 cells. The amountof IFN-α present in culture supernatants of porcine peripheral bloodmononuclear cells (PBMC) exposed for 18 h to the indicated PRRS virusstock in the presence or absence of TGE virus was determined by ELISA.na=not applicable; *Response to TGEv alone.

Table 4 shows decreased interferon-α inhibitory effect of the P129-PK-FLand P129-PK-d43/44 viruses as compared to the wild-type P129 virus andthe PRRS Ingelvac vaccines. The amount of IFN-α present in culturesupernatants of porcine peripheral blood mononuclear cells (PBMC)exposed for 18 h to the indicated PRRS virus stock in the presence orabsence of TGE virus was determined by ELISA. na=not applicable;*Response to TGEV alone.

The plentiful amount of IFN-α secreted by PBMC in response to theirexposure to TGEV is derived primarily from a subset of cells thatcomprise less than 0.3% of the PBMC population. This infrequent butimportant cell subset is composed of plasmacytoid dendritic cells(PDCs), which received this name due to characteristic plasmacytoidmorphology. To further examine the IFN-α phenotype of the P129-PK-FL andthe P129-PK-d43/44 viruses, a series of experiments was performedsimilar to those described above, except that PDC freshly isolated fromPBMC to a >95% purity were utilized. As shown in FIG. 1, this series ofexperiments confirmed that the P129-PK-FL and P129-PK-d43/44 virusescaused negligible inhibition of IFN-α induction by TGEV. Furthermore, inone experiment an apparent enhancing effect was observed onTGEV-mediated IFN-α induction by PDCs in response to P129-PK-FL andP129-PK-d43/44 PRRS viruses. In contrast, the Ingelvac PRRS MLV virusexhibited a strong inhibitory effect on the IFN-α response, as shown inFIG. 1.

The results described in the experimental section reveal that theP129-PK-FL and P129-PK-d43/44 PRRS viruses, as well as other derivativesof the pCMV-S-P129-PK17-FL infectious cDNA clone, have a greatly reducedability to inhibit the induction of IFN-α by TGEV in infected PBMC orPDC cells. This is in marked contrast to the IFN suppressive effectobserved with wild-type (low-passage) PRRS viruses and with twocommercially available modified live virus vaccines (Ingelvac PRRS MLVand Ingelvac PRRS ATP). The observation that the P129-PK-FL andP129-PK-d43/44 viruses were minimally suppressive of this importantfunction of PDCs is potentially significant given the major role thatthese cells play in mediating innate immunity against virus infections.

It should also be noted that the present invention provides clinicallyeffective commercial vaccine viruses adapted to grow on permissive cellsthat recombinantly express CD163 receptor, and that such viruses andvaccines are not dependent on, nor were developed at any point with,historical “simian cell” culturing technology. See specifically U.S.Pat. No. 7,754,464.

Example 6 Safety and efficacy of vacclne candidates

In order to evaluate their safety and efficacy as vaccines against PRRS,three of the viruses derived from the pCMV-S-P129-PK17-FL infectiouscDNA clone, P129-PK-FL (passage 7/24), P129-PK-d43/44 (passage 17/34)and P129-PK-d43/44/46 (passage 17/34) were evaluated in a young pigrespiratory disease model. The origin of these viruses is shown in FIG.8, and the experimental design (treatment groups) are listed in Table 5.Low passage virulent PRRSV isolate NADC20 was used for heterologouschallenge at 7 weeks of age (four week past vaccination). Controltreatment groups included mock vaccine and commercial PRRS vaccineIngelvac MLV.

Non-treated (NT) groups were as follows: NT1 pigs were sentinels tomonitor the health status of source pigs. They were housed separatelyand were necropsied prior to PRRSV challenge. NT2 pigs were contactcontrols housed separately in a pen between the two pens of vaccinatedpigs, a total of two per treatment group for each T02 thru T05. NT3 pigswere contact controls housed one per pen with vaccinated pigs, a totalof two per treatment group (T01 thru T05). Only NT3 pigs were assignedto the T01 group.

Rectal temperatures of vaccinated animals were measured post-1460vaccination and compared to the T01 (mock vaccine) treatment group. Theresults are shown in FIG. 1. None of the vaccines induced fevers. Allgroups averaged less than 104° C. throughout the post-vaccinationobservation period.

Rectal temperatures of pigs were measured post-challenge. The resultsare shown in FIG. 2. Unvaccinated T01 pigs showed sustained fevers ofgreater than 104° C. In contrast, three of the vaccines significantlyreduced post-challenge fevers. P129-PK-FL was most effective at reducingfevers.

The body weight of animals was recorded pre- and post-vaccination. Theresults are shown in FIG. 3. Body weights were recorded on days −1(prior to vaccination), 10, 24, 27 (prior to challenge), and 37 of thestudy. In unvaccinated pigs, challenge with virulent NADC20 virus almostcompletely eliminated weight gain during the 10 day observation period.The vaccines negated this effect to various degrees.

The lungs of challenged animals were examined at necropsiedpost-challenge. The percentages of each lung involved in lesions areshown in FIG. 4. The T01 mock vaccine group averaged 25.1% lung lesioninvolvement. The vaccines reduced lung lesions to various degrees.P129-PK-FL was most efficacious, reducing lung lesion involvement to1.1%.

The severity of the lung lesions was evaluated using a lung assessmentscore (LAS) as shown in FIG. 5. Three of the vaccines reduced LAS.P129-PK-1480 FL reduced the mean LAS from 1.63 in the mock vaccinatedgroup to 0.14.

Serum antibodies to PRRSV were induced by both vaccination andchallenge. The IDEXX ELISA S/P ratios were measured on days 27 and 37 ofthe study. The results are shown in FIG. 6. Vaccination with P129-PK-FLinduced the highest levels of anti-PRRS antibodies.

Viremia in the serum of challenged pigs was titrated on PAM cells.Results (TCID₅₀/mL) are given in FIG. 7. P129-PK-FL was most effectiveat reducing post-challenge viremia.

Although the invention has been described with reference to the aboveexamples, and to Attachments, the entire content of each of which areincorporated by reference in their entireties, it will be understoodthat modifications and variations are encompassed within the spirit andscope of the invention. Accordingly, the invention is limited only bythe following claims.

Example 7 Derivation of Infectious cDNA Clone pCMV-S-P129-PK17-FL fromInfectious cDNA Clone pCMV-S-P129

The PRRSV infectious cDNA clone of the present inventionpCMV-S-P129-1500 PK17-FL can readily be derived from the previouslydescribed PRRSV infectious cDNA clone pCMV-S-P129 by one of ordinaryskill in the art, using the technique of site-directed mutagenesis.PRRSV infectious cDNA clone pCMV-S-P129 is described in U.S. Pat. No.6,500,662 and deposited with ATCC under accession number 203489. The DNAsequence of the PRRSV genome in this clone also available in the Genbank(NCBI) database as accession number AF494042. Site-directed mutagenesiskits are commercially available from a number of suppliers, and arecapable of making numerous simultaneous nucleotide changes at multiplesites in large plasmids. Such kits include, but are not limited to,Change-IT™ Multiple Mutation Site Directed Mutagenesis Kit(Affymetrix/USB), QuikChange Lightning Multi Site-Directed MutagenesisKit (Agilent Technologies-Stratagene Products), and AMAP MultiSite-directed Mutagenesis Kit (MBL International).

A list of nucleotide changes between PRRSV infectious cDNA clonepCMV-S-P129 (available from ATCC) and the PRRSV infectious cDNA clone ofthe present invention pCMV-S-P129-PK17-FL is presented in Table 8. Allchanges are in the protein coding regions of the genome. There are atotal of 74 nucleotide changes, which can be introduced into thepCMV-S-P129 infectious clone using 74 mutagenic primers and multiplesequential reactions with a commercial site-directed mutagenesis kit,yielding a plasmid molecule that is identical in sequence topCMV-S-P129-PK17-FL described herein. In actuality, one can get the sameresult with fewer than 74 mutagenic primers, since clusters of mutationswithin about 50-60 nucleotides of each other can be changed using asingle mutagenic primer. For example, nucleotides 735, 750, and 756 canbe changed using a single mutagenic primer, as can nucleotides 965, 992,and 1009. Thus the number of primers is reduced to about 60.

Of the 74 nucleotide changes, the majority (42) are synonymous or“silent”, meaning they encode the same amino acid. These nucleotidechanges are unlikely to have any measurable effect on the interferoninduction or inhibition phenotype of the virus. The remaining 32nucleotide changes are non-synonymous or “non-silent”, and result inamino acid changes in viral proteins. These 32 nucleotide changes arepredicted to be directly responsible for the interferoninduction/inhibition phenotype of the virus, and should be changed inorder to convert the virus encoded by the infectious clone pCMV-S-P129to the same interferon phenotype as the shown by the virus encoded byinfectious clone pCMV-S-P129-PK17-FL. Such a change would require atmost 32 mutagenic primers, less if one takes into account the clusteringof some of the relevant nucleotides.

Example 8 De Novo Synthesis of Infectious cDNA Clone pCMV-S-P129-PK17-FL

As an alternative to site-directed mutagenesis, the PRRS viral genome ofthe present invention can be chemically synthesized de novo, withappropriate 5′ and 3′ adaptor sequences, and cloned into the plasmidbackbone used for PRRS infectious cDNA clone pCMV-S-P129 (available fromATCC as accession number 203489) or a similar plasmid backbone. Customsynthesis of genes greater than 50 kb in length (the PRRSV genome isabout 15.5 kb) is available as a commercial service from numerousvendors, including (but not limited to): GenScript, DNA 2.0, and BioBasic Inc. The synthetic viral genome is directionally cloned into thepCMV-S vector by replacing the viral genome in the infectious clonepCMV-S-P129 using the 5′ PacI and 3′ SpeI restriction enzyme sites thatflank the genome. In order to cut the synthetic genome, a 24-nucleotideextension (5′-GCAGAGCTCGTTAATTAAACCGTC-genome-3′, which includes theunderlined PacI site) is built into the 5′ end of the synthetic genome,and an 83-nucleotide extension(5′-genome-AAAAAAAAAAAAAAAAAAAAATGCATATTTAAATCCCAAGCCGAATTCCAGCACACTGGCGGCCGTTACTAGTGAGCGGCCGC-3′, which includes the underlined SpeIsite) is built into the 3′ end of the synthetic genome. After cuttingthe plasmid and synthetic genome with PacI and SpeI, the appropriatefragments are purified, joined using DNA ligase, and transformed intoEscherichia coli for screening and propagation using standard cloningtechniques well known to persons of ordinary skill in the art.

TABLE 1 Infectious clone Virus pCMV-S-P129-PK17-FL P129-PK-FLpCMV-S-P129-PK17-d43/44 P129-PK-d43/44 pCMV-S-P129-PK17-d43/44/46P129-PK-d43/44/46 pCMV-S-P129-PK17-nsp2 P129-PK-nsp2pCMV-S-P129-PK17-nsp2-d43/44/46 P129-PK-nsp2-d43/44pCMV-S-P129-PK17-nsp2-d43/44/46 P129-PK-nsp2-d43/44/46

TABLE 2 PRRS virus titer IFN-α Estimated (TCID₅₀) (ng/ml) IFN-α PRRS inPRRS produced (ng/ml) virus virus by PBMC produced titer stocks in byPBMC Inhibition (TCID₅₀) generated response in of IFN-α Passage/ in into response response PRRSv Cell original ZMAC-1 PRRSv to PRSSv + to TGEvSAMPLE stock CLONE type stock cells alone TGEv (%) 1 P3412 wt 0 (serum)nd 10⁵ <0.04 <0.04 99 2 P3412 A 41/PK 10³ 10³ <0.04 1.166 93 3 P3412 C41/PK 10³ 10³ 0.464 3.376 81 4 P3412 A 43/FK 10⁵ 10⁶ <0.04 <0.04 99 5P3412 B 43/FK 10⁵ 10⁵ <0.04 1.86 89 6 P129 wt 0 (serum) nd 10⁷ <0.040.327 98 7 P129 A 63/PK 10⁴ 10⁸ <0.04 <0.04 99 8 P129 B 63/PK 10⁴ 10⁸<0.04 2.3 87 9 P129 A 60/FK 10⁵ 10⁶ 0.05 2.151 88 10  P129 B 60/FK 10⁵10⁶ <0.04 <0.04 99 11  P129 A-1 51/MARC 10⁵ 10⁸ <0.04 0.686 96 12  P129A-1 151/MARC 10⁵ 10⁸ <0.04 0.04 99 13  NADC wt 0 (serum) nd 10⁷ <0.04<0.04 99 20 14  IND5 wt 0 (serum) nd 10⁷ <0.04 <0.04 99 — Mock — — na na0 17.54* na

TABLE 3 IFN-α IFN-α (ng/ml) (ng/ml) Inhibition produced by produced byof TGEv PBMC in PBMC in induced response to response to IFN-α PRRSvPRRSv + response Experiment PRRS virus stock alone TGEv (%) 1 P129 0.090.64 95 P129-PK-FL 0.53 7.37 38 P129-PK-d43/44 0.85 8.59 28 P129-PK-d43/1.01 7.65 36 44/46 P129-PK- 1.34 10.28 24 dnsp2d43/44 P129-PK- 0.38 8.2831 dnsp2d43/44/46 P129-PK-dnsp2 0.04 4.86 60 Mock na 12.16* na

TABLE 4 IFN-α IFN-α (ng/ml) (ng/ml) Inhibition produced by produced byof TGEv PBMC in PBMC in induced response to response to IFN-α PRRSvPRRSv + response Experiment PRRS virus stock alone TGEv (%) 1 P129 0.049.75 81 P129-PK-FL 0.07 60.22 0 P129-PK-d43/44 0.13 70.10 0 IngelvacPRRS 0.04 13.05 74 Ingelvac PRRS 0.04 9.10 82 ATP NVSL-14 0.01 13.35 74Mock na 50.06* na 2 P129-PK-FL 0.126 13.52 26 P129-PK-d43/44 0.127 17.186 Ingelvac PRRS 0.04 3.09 83 Ingelvac PRRS 0.04 3.11 83 ATP Mock na18.21* na 3 P129-PK-FL 0.04 8.28 6 P129-PK-d43/44 0.06 8.26 6 IngelvacPRRS 0.04 3.56 60 Ingelvac PRRS 0.04 4.15 53 ATP Mock na 8.84* na 4P129-PK-FL 0.05 12.97 7 P129-PK-d43/44 0.05 13.57 3 Ingelvac PRRS 0.046.07 57 Ingelvac PRRS 0.04 5.30 62 ATP Mock na 13.95* na

TABLE 5 Volume Passage per IM # of TX IVP or Serial # Cell Line RegimenDose Pigs NT1* NA NA NA NA NA 3 NT2* NA NA NA NA NA 8 NT3* NA NA NA NANA 10 T01 Mock NA NA Day 0 2 mL 12 T02 P129-PK- 17/34 PK9 Day 0 2 mL 12d43/44 T03 P129-PK- 17/34 PK9 Day 0 2 mL 12 d43/44/46 T04 P129-PK-FL 7/24 PK9 Day 0 2 mL 12 T05 BI (Ingelvac NA Monkey Day 0 2 mL 12 MLV)kidney

TABLE 6 Passage Passage Affected Amino Passage 17 Genome Passage 0 17viral acid 0 amino amino position nucleotide nucleotide protein positionacid acid 407 C T Nsp1a 72 P P 612 C T Nsp1a 141 H Y 735 G A Nsp1b 2 D N750 A G Nsp1b 7 S G 756 G A Nsp1b 9 D N 992 A T Nsp1b 87 E D 1009 G ANsp1b 93 C Y 1096 C A Nsp1b 122 P H 1215 C T Nsp1b 162 L L 1620 G A Nsp294 A T 1786 C A Nsp2 149 T K 1793 C T Nsp2 151 G G 1808 T C Nsp2 156 D D1841 C T Nsp2 167 C C 2106 A G Nsp2 256 I V 2164 T G Nsp2 275 M R 2185 TC Nsp2 282 M T 2318 A G Nsp2 326 S S 2403 G T Nsp2 355 V L 2591 G A Nsp2417 L L 2804 C T Nsp2 488 D D 3019 A G Nsp2 560 Y C 3074 T C Nsp2 578 SS 3167 C T Nsp2 609 D D 3214 G A Nsp2 625 R K 3563 C T Nsp2 741 I I 3740A G Nsp2 800 A A 4154 T C Nsp2 938 C C 4477 A C Nsp3 72 K T 4643 A GNsp3 127 V V 4705 T C Nsp3 148 V A 4736 C T Nsp3 158 P P 5231 C T Nsp3323 I I 5324 C T Nsp3 354 L L 5393 G A Nsp3 377 L L 5498 A G Nsp3 412 LL 5851 C A Nsp4 84 A E 5855 T C Nsp4 85 D D 5909 C T Nsp4 103 V V 5917 GA Nsp4 106 S N 5985 C A Nsp4 129 L I 6505 C T Nsp5 98 A V 6644 T C Nsp5144 F F 6653 T C Nsp5 147 R R 7419 G A Nsp7 217 D N 8032 A G Nsp9 162 AA 8074 C T Nsp9 176 G G 8200 A G Nsp9 218 G G 8593 T C Nsp9 349 P P 8831G A Nsp9 429 V I 8911 T C Nsp9 455 N N 9160 A G Nsp9 538 E E 9568 A GNsp9 674 L L 9714 T C Nsp10 38 I T 10,271 C T Nsp10 224 L L 10,627 T CNsp10 342 V V 11,265 G A Nsp11 114 G E 11,512 T C Nsp11 196 S S 11,913 TC Nsp12 107 I T 12,487 A T ORF2a 144 E V 12,876 C T ORF3 66 A V 12,949 AG ORF3 90 L L 13,575 G A ORF4 117 V V 13,857 T G ORF5 29 V G 13,869 A GORF5 33 N S 13,872 C G ORF5 34 T S 14,102 G A ORF5 111 V I 14,143 C TORF5 124 V V 14,257 G A ORF5 162 E E 14,287 C T ORF5 172 N N 14,379 T CORF6 7 D D 14,546 T C ORF6 63 V A 14,578 A G ORF6 74 T A 14,780 C T ORF6141 T I 14,932 T C ORF7 20 N N 14,973 G A ORF7 34 S N 15,124 T C ORF7 84N N 15,288 G A 3′UTR — — —

TABLE 7 Nucleotide Amino acid Non-conservative ORF or nsp differencesdifferences amino acid differences Nsp1a 2 1 1 Nsp1b 7 6 5 Nsp2 19 8 5Nsp3 8 2 1 Nsp4 5 3 2 Nsp5 3 1 0 Nsp6 0 0 0 Nsp7 1 1 1 Nsp8 0 0 0 Nsp9 81 0 Nsp10 3 1 1 Nsp11 2 1 1 Nsp12 1 1 1 ORF2a 1 1 1 ORF2b 0 0 0 ORF3 2 10 ORF4 1 0 0 ORF5 7 4 2 ORF6 4 3 2 ORF7 3 1 1

TABLE 8 Passage 10 (on Passage Passage MARC- 17 (on 10 on Passage 145PK-9 MARC- 17 on PK- Affected Amino cells) cells) Genome 145 cells 9cells viral acid amino amino position nucleotide nucleotide proteinposition acid acid 407 C T Nsp1a 72 P P 612 C T Nsp1a 141 H Y 735 G ANsp1b 2 D N 750 A G Nsp1b 7 S G 756 G A Nsp1b 9 D N 965 T C Nsp1b 78 G G992 A T Nsp1b 87 E D 1009 G A Nsp1b 93 C Y 1096 C A Nsp1b 122 P H 1215 CT Nsp1b 162 L L 1376 A G Nsp2 12 A A 1395 T C Nsp2 19 C R 1871 A G Nsp2177 L L 2185 T C Nsp2 282 M T 2235 G A Nsp2 299 D N 2403 G T Nsp2 355 VL 2731 A G Nsp2 464 Y C 2804 C T Nsp2 488 D D 2918 T C Nsp2 526 S S 3019A G Nsp2 560 Y C 3067 G A Nsp2 576 G E 3074 T C Nsp2 578 S S 3214 G ANsp2 625 R K 3256 T C Nsp2 639 L S 3563 C T Nsp2 741 I I 3740 A G Nsp2800 A A 4154 T C Nsp2 938 C C 4477 A C Nsp3 72 K T 4643 A G Nsp3 127 V V4705 T C Nsp3 148 V A 4736 C T Nsp3 158 P P 4784 C T Nsp3 174 V V 5231 CT Nsp3 323 I I 5324 C T Nsp3 354 L L 5498 A G Nsp3 412 L L 5855 T C Nsp485 D D 5862 G A Nsp4 88 A T 5985 C A Nsp4 129 L I 6155 T C Nsp4 185 D D6505 C T Nsp5 98 A V 6776 A G Nsp7 2 L L 7419 G A Nsp7 217 D N 7521 T CNsp7 251 S P 8032 A G Nsp9 162 A A 8074 C T Nsp9 176 G G 8200 A G Nsp9218 G G 8263 T C Nsp9 239 S S 8485 T C Nsp9 313 H H 8593 T C Nsp9 349 PP 8831 G A Nsp9 429 V I 8911 T C Nsp9 455 N N 9022 A G Nsp9 492 L L 9714T C Nsp10 38 I T 9934 C T Nsp10 111 N N 10,237 G A Nsp10 212 L L 10,271C T Nsp10 224 L L 10,333 T C Nsp10 244 L L 10,520 A G Nsp10 307 M V10,627 T C Nsp10 342 V V 10,847 A C Nsp10 416 I L 10,867 C T Nsp10 422 FF 10,936 C T Nsp11 4 S S 11,512 T C Nsp11 196 S S 12,949 A G ORF3 90 L L13,452 C T ORF4 76 P P 13,575 G A ORF4 117 V V 13,843 T G ORF5 24 C W13,860 G A ORF5 30 S N 14,287 C T ORF5 172 N N 14,481 A G ORF6 41 L L14,546 T C ORF6 63 V A 14,578 A G ORF6 74 T A 14,780 C T ORF6 141 T I14,932 T C ORF7 20 N N

TABLE 9 Amino acid changes responsible for reduced inter-  feron inhibition and attenuation of virulence. Genome Nsp and PassagePassage Passage Position ORF and position 0 amino 17 amino 52 amino (nt)position(aa) (aa) acid acid acid    735 ORF1a: 182 Nsp1b: 2 AMADVYDAMANVYD AMANVYD    756 ORF1a: 189 Nsp1b: 9 ISHDAVM IGHNAVM IGHNAVM   992 ORF1a: 267 Nsp1b: 87 TVPEGNC TVPDGNC TVPDGNC   1009 ORF1a: 273Nsp1b: 93 CWWCLFD CWWYLFD CWWYLFD   1096 ORF1a: 302 Nsp1b: HGVPGKYHGVHGKY HGVHGKY 122   2106 ORF1a: 639 Nsp2: 256 AAKIDQY AAKVDQY AAKVDQY  2185 ORF1a: 665 Nsp2: 282 PSAMDTS PSATDTS PSATDTS   2403 ORF1a: 738Nsp2: 355 LVSVLSK LNSLLSK LNSLLSK   3019 ORF1a: 943 Nsp2: 560 APMYQDEAPMCQDE APMCQDE   4477 ORF1a: 1429 Nsp3: 72 CAPKGMD CAPTGMD CAPTGMD  4705 ORF1a: 1505 Nsp3: 148 PKVVKVS PKVAKVS PKVAKVS   5985 ORF1a: 1932Nsp4: 129 AGELVGV AGEIVGV AGEIVGV   7419 ORF1a: 2410 Nsp7: 217 ADFDPEKADFNPEK ADFNPEK   8831 ORF1a/1b: 2881 Nsp9: 429 QTPVLGR QTPILGR QTPILGR13,857 ORF5: 29 — AVLVNAN AVLGNAN AVLGNAN 14,578 ORF6: 74 — VALTMGAVALAMGA VALAMGA 14,780 ORF6: 141 — PGSTTVN PGSITVN PGSITVN

TABLE 10 Amino acid changes responsible for reduced inter-feron inhibition and attenuation of virulence Genome ORF and Nsp andPassage Passage Passage Position position position 0 amino  17 amino52 amino (nt) (aa) (aa) acid acid acid  735 ORF1a: 182 Nsp1b: 2 AMADVYDAMANVYD AMANVYD  756 ORF1a: 189 Nsp1b: 9 ISHDAVM IGHNAVM IGHNAVM 1009ORF1a: 273 Nsp1b: 93 CWWCLFD CWWYLFD CWWYLFD 1096 ORF1a: 302 Nsp1b:HGVPGKY HGVHGKY HGVHGKY 122 2185 ORF1a: 665 Nsp2: 282 PSAMDTS PSATDTSPSATDTS 3019 ORF1a: 943 Nsp2: 560 APMYQDE APMCQDE APMCQDE 4477ORF1a: 1429 Nsp3: 72 CAPKGMD CAPTGMD CAPTGMD 4705 ORF1a: 1505 Nsp3: 148PKVVKVS PKVAKVS PKVAKVS 7419 ORF1a: 2410 Nsp7: 217 ADFDPEK ADFNPEKADFNPEK

Example 9 Vaccine Consisting of P129-PKC12-FL Virus is Safe for Use in 1Day Old Pigs and Efficacious for at Least 26 Weeks

Existing PRRS modified live vaccines are only recommended for use inpigs two weeks of age or older. Studies were conducted to determine ifthe attenuated P129-PKC12-FL virus at passage 57 (2.13 log₁₀ TCID₅₀ in a2 mL dose) is safe for use in 1-day old neonatal pigs, and sufficientlyimmunogenic to provide protection against a virulent heterogeneouschallenge at up to 6 months (26 weeks) following vaccination. (this isessentially the same as the passage 52 virus, as described in SEQ ID No:6).

A total of 22 of 24 PRRSV seronegative pregnant sows sourced producedhealthy piglets. These pigs (piglets) were administered a single 2.0 mLdose of the Mock Vaccine or the P129-PKC12-FL virus vaccine as anintramuscular injection at approximately 1 day of age (Day 0) accordingto an allotment. All healthy piglets in a litter/farrowing cratereceived the same Mock Vaccine (11 litters, 100 piglets) orP129-PKC12-FL virus vaccine (11 litters, 91 piglets) on the same day.Mock vaccinated sows and piglets remained PRRSV negative throughout thevaccination period. No confounding disease factors were detected. Theprimary variables used to demonstrate safety in piglets vaccinated at 1day of age were clinical observations post-vaccination. Serology wasused to confirm successful vaccination of all piglets.

Clinical observations were observed and recorded for all piglets on Days1 thru 10 post vaccination. Of the 100 pigs (piglets) administered thecontrol product (T01) and 91 piglets administered the test product(T02), 15 and 14 pigs, respectively were observed to be not normal(Table 11, referring to the tables as numbered within this specificexample). Piglets observed as not normal were further noted as havingabnormal general condition and/or depression.

TABLE 11 Number of Pigs Ever Observed with Clinical Signs Following aVaccination at One Day of Age with a Modified Live PRRSV Vaccine orControl [Number of animals observed with an abnormal health condition (%animals observed with an abnormal condition)] Treatment Not GeneralRespiratory Group Normal Condition Depression Distress Cough SneezeOther Mock 15 (15.3) 15 (15.3) 5 (5.2) 0 0 0 0 Vaccine P129- 14 (15.4)14 (15.4) 5 (5.6) 0 0 0 0 PKC12- FL Vaccine

IDEXX ELISA results confirmed all pigs were negative for PRRSV prior tovaccination (S/P ratio<0.4) and all mock vaccine controls remainedserologically negative during the vaccination phase of this study.Following vaccination, all pigs in the P129-PKC12-FL Vaccine group weresero-positive (S/P ratio>0.4) by Day 21 or 22 (Table 12 below).

TABLE 12 Serum Geometric Mean PRRSV Titers (IDEXX Elisa) Following aVaccination at One Day of Age with a Modified Live PRRSV Vaccine orControl [Mean titer (Animals positive*/total animals)] Treatment GroupDay 0 Day 10/11 Day 21/22 Mock Vaccine 0.002 (0/100) 0.000 (0/86)  0.001(0/85)  P129-PKC12-FL 0.002 (0/91)  1.322 (76/85) 2.412 (81/81) Vaccine

The data supports the conclusion that attenuated P129-PKC12-FL virus atpassage 57 is safe when administered as a single 2 mL IM dose to pigletsproduced by seronegative sows at one day of age when observed thoughweaning at 21 or 22 days of age.

Prior to challenge, mock vaccinated and P129-PKC12-FL virus vaccinatedpiglets were re-housed in pens (2 piglets from the same vaccinationgroup per pen) in rooms, such that each room contained 12 pens from eachvaccination group. Challenged was with the virulent heterologous PRRSisolate NADC20 at either 7, 18, or 26 weeks post-vaccination. Achallenge dose equaled 4.0 mL (1.0 mL per nostril plus a 2.0 mLintramuscular injection) of NADC20 stock solution at 2.27 log₁₀TCID₅₀/mL (2.87 log_(in) TCID₅₀/4 mL dose). NADC-20 is a very virulentgenotype 2 PRRS virus, provided by Dr. Kelly Lager of the NationalAnimal Disease Center, USDA (Ames, Iowa). The NADC-20 ORF5 amino acidsequence is 94.5% identical to P129-PKC12-FL.

The primary variable in determining prevention of disease was percentlung with lesions. Lung lesions were scored at necropsy (10 dayspost-challenge) such that the percentage of consolidation for each lobe(left cranial, left middle, left caudal, right cranial, right middle,right caudal, and accessory) were scored and recorded as percent of lobeobserved with lesions.

At 7 weeks post-vaccination, percent lung with lesions was significantlygreater in the mock vaccinated pigs compared to P129-PKC12-FL virusvaccinated piglets (P≦.0001) (Table 13).

TABLE 13 Back Transformed Least Square Means Percent Lung with LesionsFollowing a PRRSV NADC20 Challenge of 7-Week-Old Piglets PreviouslyVaccinated at One-Day of Age with a Modified Live PRRSV Vaccine or MockVaccine Lower Upper 95% 95% Treatment Number % Lung Standard ConfidenceConfidence Group of Pigs with Lesions Error Interval Interval Range Mock24 43.9 3.48 36.3 51.7 18.5-68.5 Vaccine P129- 22 0.7 0.29 0.2 1.5  0-8.25 PKC12-FL Vaccine

At 18 weeks post-vaccination, percent lung with lesions wassignificantly greater in the mock vaccinated piglets compared toP129-PKC12-FL virus vaccinated piglets (P≦.0001) (Table 14).

TABLE 14 Back Transformed Least Square Means Percent Lung with LesionsFollowing a PRRSV NADC20 Challenge of 18-Week-Old Piglets PreviouslyVaccinated at One-Day of Age with a Modified Live PRRSV Vaccine orControl Lower Upper 95% 95% Treatment Number % Lung Standard ConfidenceConfidence Group of Pigs with Lesions Error Interval Interval Range Mock23 21.1 3.70 13.6 29.7 1.9-63.0 Vaccine P129- 20 1.0 0.43 0.3 2.1  0-5.95 PKC12-FL Vaccine

At 26 weeks post-vaccination, percent lung with lesions wassignificantly greater in the mock vaccinated pigs compared toP129-PKC12-FL virus vaccinated piglets (P≦.0001) (Table 15).

TABLE 15 Back Transformed Least Square Means Percent Lung with LesionsFollowing a PRRSV NADC20 Challenge of 26-Week-Old Piglets PreviouslyVaccinated at One-Day of Age with a Modified Live PRRSV Vaccine orControl Lower Upper 95% 95% Treatment Number % Lung Standard ConfidenceConfidence Group of Pigs with Lesions Error Interval Interval Range Mock24 17.7 2.59 12.4 23.8 4.5-51 Vaccine P129- 24 1.2 0.72 0.1 3.3   0-20PKC12-FL Vaccine

The results indicate that attenuated vaccine virus P129-PKC12-FL is safein 1-day old piglets, and is capable of inducing a potent immunologicalresponse with an exceptional duration of immunity. A single doseprotects 1-day old piglets from a virulent heterologous PRRS challengefor at least 26 weeks.

These properties of safety in 1-day old piglets and 26 week duration ofPRRS immunity is useful for multivalent combination swine vaccines, suchas bivalent PRRSV/Mycoplasma hyopneumoniae (M.hyo) vaccines, bivalentPRRSV/Porcine Circovirus type 2 (PCV2) vaccines, and trivalentPRRSV/M.hyo/PCV2 vaccines, as well as for monovalent PRRSV vaccines.

Example 10 Vaccine Consisting of P129-PKC12-FL Virus Provides an EarlyOnset of Protective Immunity

Existing modified live PRRS vaccines are recommended for vaccination atleast 3 to 4 weeks prior to exposure to virulent PRRS strains. This timeinterval is believed to be necessary in order to establish protectiveimmunity. The study described here demonstrates significant protectionagainst a virulent heterologous PRRS virus challenge delivered only 14days following vaccination with the P129-1705 PKC12-FL virus vaccine andtwo other commercial PRRS vaccines.

During the vaccination phase, treatment groups of 18 pigs (at 3 weeks ofage) were housed in four separate rooms in pens of 6 animals each. Pigs(piglets) were administered a single intramuscular injection of the MockVaccine (2 mL), the attenuated P129-PKC12-FL passage 57 virus vaccine(3.62 log₁₀ TCID₅₀ in a 2 mL dose), Ingelvac PRRS MLV vaccine, orIngelvac PRRS ATP vaccine, at approximately 3 weeks of age (Day 0)according to manufacturer's instructions.

Prior to challenge, all remaining animals were re-housed in pens of 3animals each, with one empty pen between each occupied pen, such thatmultiple pens of animals from each treatment group were housed in eachof four rooms. Challenged was with the virulent heterologous PRRSisolate NADC20 at approximately 5 weeks of age (Day 14). A challengedose equaled 4.0 mL (1.0 mL per nostril plus a 2.0 mL intramuscularinjection) of NADC20 stock solution at 2.07 log₁₀ TCID₅₀/mL (2.67 log₁₀TCID₅₀/4 mL dose).

The primary variable in determining reduction of disease was percentlung lesions in the vaccinated groups in relation to mock vaccine group.Significant differences were found between all vaccinated groups(P129-PKC12-FL, P=0.0177; Ingelvac PRRS ATP, P=0.0255; Ingelvac PRRSMLV, P=0.0137) when compared to the mock vaccinated group. Nosignificant differences were found when comparing vaccinated groups toeach other (Table 16).

TABLE 16 Percent Lung with Lesions Following a PRRSV NADC20 Challenge ofFive-Week-Old Pigs Previously Vaccinated with a Modified Live PRRSVVaccine or Mock Vaccine. Treatment Number % Lung Group of Pigs withLesion Standard Error Range Mock Vaccine 18 46.1 10.12  1.4-88.4P129-PKC12-FL 18 17.5 7.71 0.23-76.6 Vaccine Ingelvac PRRS 18 18.9 7.96 0.18-88.96 ATP Vaccine Ingelvac PRRS 17 16.0 7.62 0.45-71.2 MLV Vaccine

These results demonstrate that it may be a general property of modifiedlive PRRS virus vaccines to induce partial immunity and a reduction ofdisease by 14 days post-vaccination. This property may result from acombination of early acquired immunity (e.g. specific antibodies andcytotoxic T cells), innate immunity (e.g. induced interferons andnatural killer cells), and/or competition between the vaccine virus andthe challenge virus for limited numbers of permissive host cells (e.g.alveolar macrophages) in the pig (piglet). Regardless of themechanism(s), this property can be utilized to protect pigs from diseaseassociated with natural or intentional PRRS infection (such asintentional exposure of incoming replacement gilts with an endemic farmstrain of virulent PRRS).

Thus, as aforementioned, this property of early onset of PRRS immunityis useful for multivalent combination swine vaccines, such as bivalentPRRSV/Mycoplasma hyopneumoniae (M.hyo) vaccines, bivalent PRRSV/PorcineCircovirus type 2 (PCV2) vaccines, and trivalent PRRSV/M.hyo/PCV2vaccines, as well as for monovalent PRRSV vaccines. As to the componentsof such vaccines, useful in the practice of the present invention (i.e.to provide early and safe vaccination as early as when the piglet is 1day of age, optionally with onset of immunity at two weeks thereafter),reference is made to all the combination vaccine components as describedin U.S. provisional application 61/620,189 of Niztel et al, entitled“PCV/Mycoplasma hyopneumoniae/PRRSZ Combination Vaccine”, filed Apr. 4,2012, the complete and entire disclosure of which is incorporated byreference herein, as if set forth in its entirety.

In regard of specific PRRS vaccines (or PRRS vaccine strains) that maybe used in the practice of the present invention (i.e. to provide earlyand safe vaccination as early as when the piglet is 1 day of age,optionally with onset of immunity at two weeks thereafter), attention isdirected to Table 1 of Murtaugh et al., Vaccine, vol 29, pp. 8192-8204,(2011), see Page 8196 thereof, where numerous such viruses/vaccines areidentified, including, without limitation, Ingelvac PRRS MLV, IngelvacPRRS ATP, and Suvaxyn PRRS (derived from Iowa State strain ISU-55). Itshould be noted that vaccines providing the above-mentioned performancecharacteristics are also expected to provide a duration of immunityperiod of about 6 months.

DEPOSIT OF BIOLOGICAL MATERIALS

The following biological materials (see also U.S. Pat. No. 6,500,662)were deposited with the American Type Culture Collection (ATCC) at 10801University Blvd., Manassas, Va., 20110-2209, USA on Nov. 19, 1998, andwere assigned the following accession numbers.

Plasmid pT7P129A, accession number 203488

Plasmid pCMV-S-P129, accession number 203489

The complete text and disclosure of the following United States Patentsis incorporated herein by reference, as if fully set forth: U.S. Pat.No. 6,500,662, and U.S. Pat. No. 7,618,797.

1. A vaccine for protecting a porcine animal against infection by a PRRSvirus, which vaccine comprises (a) a North American PRRS virus encodedby the polynucleotide molecule of SEQ ID NO: 6 or any polynucleotidewhich hybridizes thereto under highly stringent conditions,hybridization to filter bound DNA in 0.5 M NaHPO₄. 7% SDS, 1 mM EDTA at65 degrees C., and washing in 0.1×SSC/0.1% SDS at 68 degrees C., (b)said encoding polynucleotide molecule, (c) said polynucleotide moleculein the form of a plasmid, or (d) a viral vector comprising saidpolynucleotide molecule, wherein the PRRS virus is able to elicit aneffective immunoprotective response against infection by PRRS virus, inan amount effective to produce immunoprotection against infection, and acarrier suitable for veterinary use, and wherein said vaccine providesearly and safe vaccination as early as when the piglet is 1 day of age,and wherein said vaccine provides a duration of immunity for up to 6months.
 2. The vaccine of claim 1 wherein onset of immunity is providedbeginning at two weeks after vaccination.
 3. A method for vaccinating aporcine animal against infection by a PRRS virus, comprisingadministering said vaccine between about 12 hours after birth and 2weeks of age, wherein said vaccine comprises (a) an isolatedpolynucleotide molecule comprising a DNA sequence encoding an infectiousRNA molecule encoding a North American PRRS virus, (b) an infectious RNAmolecule encoding a North American PRRS virus; (c) said polynucleotidemolecule (a) in the form of a plasmid, or (d) a viral vector comprisingan infectious sequence.
 4. The method of claim 3 wherein protectiveimmunity arises no later than about 14 days after vaccination.
 5. Themethod of claim 4, wherein protective immunity arises at Day 15following vaccination on Day 1 of life, Day 21 following vaccination onDay 7 of life, or no later than about Day 28 following vaccination onDay 14 of life.
 6. The method of claim 3, wherein the duration ofprotective immunity provided is up to 6 months.